1 00:00:20,440 --> 00:00:24,340 Evolution of molecular detective. 2 00:00:24,340 --> 00:00:38,200 And after having talked about the broken the Stone Age and then the bronze Bronze Age, I would suggest to go to the Golden Age. 3 00:00:38,200 --> 00:00:47,330 And this is when, of course, David Shearer was invited to invade a new environment, 4 00:00:47,330 --> 00:00:58,270 a new warmer and drier environment compared to Glasgow, of course, and you can see how dry it can be here in Oxford. 5 00:00:58,270 --> 00:01:11,320 And so when I mean, here it was here at the first floor of the why we call the micro unit, which sadly has disappeared since then. 6 00:01:11,320 --> 00:01:13,840 And so that was in 1994. 7 00:01:13,840 --> 00:01:28,390 And so at that time, in 1994, they arrived in Oxford with a population of survivors of the Glasgow or the Bronze Age, I would say. 8 00:01:28,390 --> 00:01:38,020 And so namely, of course, Libya was there and Sean Collins was here today and also Gary Blakely. 9 00:01:38,020 --> 00:01:52,810 But very rapidly during that year, a new generation or a new population of future molecular detectives colonised the Oxford ecosystem. 10 00:01:52,810 --> 00:02:05,200 And so that was the first generation. Then we had the F1 generation, then we had the two, the three, the four and so on, and so many more. 11 00:02:05,200 --> 00:02:11,980 And so I apologise for those we don't have their picture on on this slide, but there are two reasons for that. 12 00:02:11,980 --> 00:02:18,790 The first reason is that I didn't have enough place to put all the people we were in Oxford at the time. 13 00:02:18,790 --> 00:02:25,240 And the second reason is that for some of them, I couldn't find a picture on the web. 14 00:02:25,240 --> 00:02:33,310 And so, so all these people were. So the competition was very tough. 15 00:02:33,310 --> 00:02:48,040 But most of these people where they were successfully selected during that selection and so many of them carried on their career as you know, 16 00:02:48,040 --> 00:02:57,880 and they spread the molecular detective agencies and they with these heritage all over the world. 17 00:02:57,880 --> 00:03:08,110 So they know many, as you may know, Molecular Detective Agency in different countries and all over the world that I would say in India, 18 00:03:08,110 --> 00:03:17,140 in a in Europe, in America and over in a small country that is somewhere in Europe that is called Belgium. 19 00:03:17,140 --> 00:03:21,640 And so this is where I am and to OK. 20 00:03:21,640 --> 00:03:33,970 And so but at the time, already that the people were in Oxford, they were, as I say, quite so successful. 21 00:03:33,970 --> 00:03:49,780 And so they were allowed to colonise a noble niche with much lower, I would say, a much lower fitness coast. 22 00:03:49,780 --> 00:03:50,680 And of course, 23 00:03:50,680 --> 00:04:02,920 you can see the difference here between the old biochemistry department and this marvellous building where we know the new biochemistry department. 24 00:04:02,920 --> 00:04:06,910 Of course, during this time there was science. 25 00:04:06,910 --> 00:04:16,630 And in fact, when they arrived in Oxford, as Marshall mentioned this morning, 26 00:04:16,630 --> 00:04:22,930 the major topic that we were working on was XDR recombination combination and XDR 27 00:04:22,930 --> 00:04:29,980 recombination is in fact a recombination that is carried out by a couple of two protein, 28 00:04:29,980 --> 00:04:35,170 SIRC and zari. And what do these protein they do? 29 00:04:35,170 --> 00:04:44,440 DNA strand exchange reaction, meaning that they cut the DNA strands and then they sort of exchange. 30 00:04:44,440 --> 00:04:55,210 They rewrite the DNA strands at the level of specific sites using a nice choreography, as you can see here, and also in the picture that is above. 31 00:04:55,210 --> 00:05:00,310 And so one intermediate important intermediate in this reaction is, of course, 32 00:05:00,310 --> 00:05:05,230 the Hollywood junction that is at the meeting at the middle of the slides. 33 00:05:05,230 --> 00:05:10,720 And this Holly the junction, as we heard today from Steve, 34 00:05:10,720 --> 00:05:19,670 was in fact the named Holly the junction because it was discovered in 1974 or 64 by Robin Holliday. 35 00:05:19,670 --> 00:05:22,880 When he was working on the homologous recombination. 36 00:05:22,880 --> 00:05:31,610 And here we are, working on site specific recombination, so I propose to call it Charrette Junction. 37 00:05:31,610 --> 00:05:39,350 And in fact, Basharat Junction is in fact very famous in Belgium. 38 00:05:39,350 --> 00:05:46,910 Everybody knows the Sherratt Junction and I would tell you why, because there is a place in Belgium that is called Chirac. 39 00:05:46,910 --> 00:05:57,240 Chirac in that place in that is there is a junction between two major motorways and in fact, way, way, way, way. 40 00:05:57,240 --> 00:06:02,390 It is so famous. It's because every day there is a traffic jam there. 41 00:06:02,390 --> 00:06:06,860 And so we heard about the shattered junction every day. 42 00:06:06,860 --> 00:06:12,710 So, so you recombination a combination as we as well. 43 00:06:12,710 --> 00:06:18,230 I would tell you as different function, I think ethics, we is going to talk about it. 44 00:06:18,230 --> 00:06:26,540 Just ask me. And so the main. Well, the first rule of XIII recombination was discovered. 45 00:06:26,540 --> 00:06:37,700 Rule was to resolve plasmid commerce. And we know we knew and we we worked on that these plasmids are controlled recombination. 46 00:06:37,700 --> 00:06:47,750 That plasmid is controlled by a mechanism that is called topological filter or mechanism that Marshall talked a little bit about this morning. 47 00:06:47,750 --> 00:06:55,010 And so this mechanism was also known since the Bruhn the Bronze Age coming from Glasgow. 48 00:06:55,010 --> 00:07:09,800 But maybe more importantly, we quite rapidly we found out that well, the detective found out not we, but detective found. 49 00:07:09,800 --> 00:07:15,800 If it seems that the chromosome sites for the resolution of chromosome military males. 50 00:07:15,800 --> 00:07:21,860 That is, this is controlled by a fantastic protein that is called F.T Escape. 51 00:07:21,860 --> 00:07:31,730 And in fact, if GSK is not just a protein that acts in XXXII recombination, it's an important cell division protein. 52 00:07:31,730 --> 00:07:50,390 And so this finding was a real turn in the detective career and all the group, I would say, because it brings David and and the agency on a new topic. 53 00:07:50,390 --> 00:07:58,520 And so instead of staying on very, quite small things, small object like plasmid transposons and Trone, 54 00:07:58,520 --> 00:08:09,440 they became interested on what happens on the wall chromosomes in bacteria and and so doing, 55 00:08:09,440 --> 00:08:18,380 because if this case not just a division protein, but it's also a protein that sort of travel along the DNA on the chromosomes. 56 00:08:18,380 --> 00:08:27,680 And so and so then the group became interested in the dynamic of chromosomes in bacteria. 57 00:08:27,680 --> 00:08:34,610 And so we're going to illustrate that first, for example, here it's the ecology cell, 58 00:08:34,610 --> 00:08:39,230 and within the cell there is the DNA, the DNA and the chromosomes and the chromosomes. 59 00:08:39,230 --> 00:08:48,470 The bacterial chromosomes has to pick for the left and right right before and since they've worked on this issue. 60 00:08:48,470 --> 00:08:56,150 We all know that the Leftwich picture is blue and the the right picture is, of course, right. 61 00:08:56,150 --> 00:08:59,150 And so this is inspired. 62 00:08:59,150 --> 00:09:10,700 Some artists, which which I'm going to show you on a video decided to illustrate the issue of the chromosome within the cells. 63 00:09:10,700 --> 00:09:18,470 So I will start the video. Maybe you've seen this video already. So the problem of the chromosome is so you have the box there. 64 00:09:18,470 --> 00:09:29,060 And one of the problem that we have to solve is how to put the chromosome because it's huge compared to the cells within a box within the cell. 65 00:09:29,060 --> 00:09:42,850 But not just that, because the replication the chromosome at some stage will replicate, so you need two chromosomes to be enter into the box. 66 00:09:42,850 --> 00:09:54,970 And so as you can see, this causes several issues that are difficult to address and also and that the group here started to address these 67 00:09:54,970 --> 00:10:02,890 issues and there are different aspect that we're still at the time there is how does the the chromosome coordinate? 68 00:10:02,890 --> 00:10:09,500 Well, know how this the the chromosome? 69 00:10:09,500 --> 00:10:18,230 Undergoes replication stop. So, OK, so well, there is a problem here. 70 00:10:18,230 --> 00:10:33,430 We're. I. I'm back. 71 00:10:33,430 --> 00:10:38,500 It's gone anyway, so my savings don't disappear. 72 00:10:38,500 --> 00:10:49,180 OK, so so so these are issues that we are going to talk about reputation, but also all the chromosome segregates. 73 00:10:49,180 --> 00:10:56,530 And of course, we're going to talk about CCR and all during all these processes is the chromosome repair. 74 00:10:56,530 --> 00:11:03,490 And so for this third session with three people, we are going to talk and the first people, 75 00:11:03,490 --> 00:11:10,210 the first guy that I will ask to come here is a fix bar from the CNRS in France. 76 00:11:10,210 --> 00:12:12,690 We will who talk about XDR recombination and various aspects of it. 77 00:12:12,690 --> 00:12:15,180 So thanks for this implication. 78 00:12:15,180 --> 00:12:24,730 So I guess I don't have much to say because I wanted to make a recap of politics here, but I think you will go to everything. 79 00:12:24,730 --> 00:12:33,480 Um, so I thought I will make a kind of. 80 00:12:33,480 --> 00:12:39,760 And so it's going to be for use in the region and. 81 00:12:39,760 --> 00:12:51,340 So the original picture. It all starts with because of the fact that we have several leprechauns in there as well. 82 00:12:51,340 --> 00:12:58,750 So it started with me, but then the genome is a bigger big secret I picked on. 83 00:12:58,750 --> 00:13:12,920 And so. I came from the east when I arrived in Lebanon, so for me, I mean, the best way to manipulate, to do a cell cycle to it would be the utility. 84 00:13:12,920 --> 00:13:17,360 So basically, you have plenty of origins. You start replicating. 85 00:13:17,360 --> 00:13:30,520 You go fast. And then once you have replicated you, you keep the two crews together with cuisines, because this way you have a problem. 86 00:13:30,520 --> 00:13:37,600 You can always save yourself homologous recombination, so we have miss these four cuisine. 87 00:13:37,600 --> 00:13:47,200 We have Steve Kay, former weekly communication. And you just decide to segregate to separate them just when you are ready to divide. 88 00:13:47,200 --> 00:13:56,120 And now the primary circle of chromosome. That's what I debated. So you what I'm drawing here is that there is no. 89 00:13:56,120 --> 00:14:04,370 Nothing, but if you just try to separate your two chromosome, you have one chance, not of two to get them tangled. 90 00:14:04,370 --> 00:14:09,320 There is no denying there is no distinction, you're just tangle like that. 91 00:14:09,320 --> 00:14:13,610 And so this is risky. 92 00:14:13,610 --> 00:14:22,130 And so maybe that's why bacteria evolve something where they decided to me, we don't want to do these kind of way before. 93 00:14:22,130 --> 00:14:29,690 I mean, it's safer to have just one origin of publication. And as soon as you do replication, you want to separate them. 94 00:14:29,690 --> 00:14:34,840 You don't run the risk of having these kind of tangled up. 95 00:14:34,840 --> 00:14:44,470 OK. And so you have to partition and then you have to separate them, so you condoms the chromosomes with collegiate conventions. 96 00:14:44,470 --> 00:14:50,630 And so in this way, you don't trust this thing. OK, so that's one problem. 97 00:14:50,630 --> 00:15:00,740 So just because of the way the cell cycle is set. There is a problem with archaea, because some archaea have a circular chromosome in three regions. 98 00:15:00,740 --> 00:15:04,750 And so this I don't understand. 99 00:15:04,750 --> 00:15:16,000 I wrote there is a second problem is the fact that DNA is it's a double Alex, so when you're implicated in the body, you have to unwind it. 100 00:15:16,000 --> 00:15:21,360 And so that's what's happening. So you have a little replication for each year. 101 00:15:21,360 --> 00:15:27,400 So I didn't draw the double helix lecture because it's too complicated for me. 102 00:15:27,400 --> 00:15:34,250 In kind of training. That's the point. 103 00:15:34,250 --> 00:15:43,400 And so when you open it, you are positive that you're insurance, they took taken care of by the judges, but Cesar Chavez is not fast enough. 104 00:15:43,400 --> 00:15:53,570 You can have the whole replication fauquier rotating so you get three cats and. 105 00:15:53,570 --> 00:15:58,560 Those precautions taken care of by to perform. 106 00:15:58,560 --> 00:16:04,570 OK, but if Cooper four is not fast enough, then you can get certain names. 107 00:16:04,570 --> 00:16:14,110 So I'm going to to put a few names on those typical things, so I'm going to introduce you. 108 00:16:14,110 --> 00:16:21,250 This is a kind of ketogenic trick trio, Proteobacteria and so the names, I'm going to introduce you. 109 00:16:21,250 --> 00:16:33,190 So because I'm very interested in your career, so your career in the Tree of Gamma, Proteobacteria is very close to equalise. 110 00:16:33,190 --> 00:16:43,060 The closest member I took on as an owl, so I'll group a to consensus. 111 00:16:43,060 --> 00:16:52,720 That's the point. And so what what what I'm going to tell you is that if you look in the in the tree, 112 00:16:52,720 --> 00:17:03,200 there is something which is common to qualifying economies that we have domesticated, omitting the transferase, which is called them. 113 00:17:03,200 --> 00:17:14,990 And we're going to move this is them. And when they domesticated them, they domesticated with it a set of genes which are always associated with them. 114 00:17:14,990 --> 00:17:26,190 And so you have psyche which binds to any immediate DNA and instead of a normal condensing the movie and we have this massive protein. 115 00:17:26,190 --> 00:17:30,970 And so all of those. Always together. 116 00:17:30,970 --> 00:17:38,530 And so now if we put this on top, we have these so is mutilated normally, 117 00:17:38,530 --> 00:17:44,890 but being the following Sunni militants, Typekit Sticky binds to it and it blocks top of my thing. 118 00:17:44,890 --> 00:17:58,480 So you have a kind of state of cohesion. Then to put up the convincing is, maybe it's not the usual type of sequencing and mapping. 119 00:17:58,480 --> 00:18:08,680 This protein is in fact specialised to keep the two region opposite of the original replication together, which is the terms. 120 00:18:08,680 --> 00:18:20,080 A difference important difference between Korea and Nicaragua that Nicolae lost as they lost the partition machinery and equalise getting equal, 121 00:18:20,080 --> 00:18:25,630 I lost as lost people, they kept it. OK, so OK. 122 00:18:25,630 --> 00:18:30,130 So now we go to chromosome dynamics and two. 123 00:18:30,130 --> 00:18:41,410 And so I just drew one single so that if that being the fork when you need to do to repair something is obviously communication. 124 00:18:41,410 --> 00:18:51,880 And so there is only one who did it. So you have the chance you should resolve it to get a crossover. 125 00:18:51,880 --> 00:18:56,510 That's fine. And so you need to solve the. 126 00:18:56,510 --> 00:18:57,230 You need to. 127 00:18:57,230 --> 00:19:07,920 You have a problem only if you are not in the middle of Kosovo, so you solve it if you have the non-member of Kosovo and this is solved by six. 128 00:19:07,920 --> 00:19:13,830 That's what so this is the machinery that burned out. 129 00:19:13,830 --> 00:19:20,480 Explain to you everything. Yeah. And next year. 130 00:19:20,480 --> 00:19:25,730 In fact, the problem is, how do you know that you have a number of Kosovo, you have to count. 131 00:19:25,730 --> 00:19:30,370 I mean, you say you have an even number of Kosovo, I don't I don't want to influence the president. 132 00:19:30,370 --> 00:19:35,960 So the way? So in fact, it doesn't guarantee just the best way to do it is to do it again. 133 00:19:35,960 --> 00:19:42,840 So you do it just before the chromosome one to separate. So you do it. 134 00:19:42,840 --> 00:19:52,740 At the time of television in determining the origin of the origin, and so at a specific site, yeah, OK. 135 00:19:52,740 --> 00:20:04,500 And so this is possible because this machinery is highly regulated and its activity depends on this huge protein, 136 00:20:04,500 --> 00:20:18,720 which is ActionScript U.S. division in which it's assembled into an anointed DNA pump and it can help position the to the to the tell me, 137 00:20:18,720 --> 00:20:23,940 is that the medicine? And then you can activate cell recognition. 138 00:20:23,940 --> 00:20:28,460 And this is very regulated because. 139 00:20:28,460 --> 00:20:34,880 The positioning of the chromosome, the poor genes on the opposite side depend on the machinery. 140 00:20:34,880 --> 00:20:38,450 It depends on the colonisation of the DNA. 141 00:20:38,450 --> 00:20:45,680 The decision to make the cell division at the appropriate position depend on nuclear integration with human, 142 00:20:45,680 --> 00:20:52,340 which is OK and then maintaining that so many swin positions together will 143 00:20:52,340 --> 00:21:02,540 depend on the matching protein and so might be all part of the same set of way. 144 00:21:02,540 --> 00:21:07,730 OK, so that's the original picture whiteboard. 145 00:21:07,730 --> 00:21:12,350 And so now that's the second part of my thought is the use of Excel. 146 00:21:12,350 --> 00:21:23,840 So I told you they are getting names when 2.4, doesn't it fast enough and getting the same, the same thing is the best way you have to. 147 00:21:23,840 --> 00:21:30,660 You want to try to resolve them at the end. And in fact, this year has been shown to be about two decades inmates. 148 00:21:30,660 --> 00:21:40,040 We can resolve dimer, but it can also be getting it on top of that because being one of the proteins have been co-opted, 149 00:21:40,040 --> 00:21:47,110 so they interact directly with folks of the proposal at the site where it needs to at. 150 00:21:47,110 --> 00:21:53,080 And so everything is goes together. OK. 151 00:21:53,080 --> 00:21:59,200 And then there's the self use of HCR, which is mobile elements. 152 00:21:59,200 --> 00:22:03,010 They said, Oh, there's this machinery inside the cell. They are handy. 153 00:22:03,010 --> 00:22:07,780 And so the Glasgow people explain everything about that. 154 00:22:07,780 --> 00:22:13,640 In fact, many of you are using Excel three of the. 155 00:22:13,640 --> 00:22:20,530 And I mean, for these two to listen to you and a class of elements which are integrated mobile elements, 156 00:22:20,530 --> 00:22:25,150 which protects you have to integrate into the deep state of the host. 157 00:22:25,150 --> 00:22:35,350 So Fiji's. So just to tell you a bit more about those elements, I have to go into the detail of it. 158 00:22:35,350 --> 00:22:42,760 So inside the Excel machinery, most back is composed of two proteins mixture of sweet tea and green and mixed, 159 00:22:42,760 --> 00:22:51,010 yielding the following along with wide dynamics and the deep side is composed of a mixture of C binding sites. 160 00:22:51,010 --> 00:22:57,580 And an excel divining site which are slightly different than the central region, 161 00:22:57,580 --> 00:23:06,010 which needs to be at the right distance so that the two proteins can interact together. 162 00:23:06,010 --> 00:23:12,650 And so. Because this is asymmetric, you have to change, in fact, you can make two types of sign ups, 163 00:23:12,650 --> 00:23:21,250 so this is two sides six weeks old bond switched sides, and so neither SLB will contact will. 164 00:23:21,250 --> 00:23:27,490 So you get this sign up for access, different tactics, different tactics you have seen since. 165 00:23:27,490 --> 00:23:32,650 And then the subsequent tactics will be in trance and the cyclic. 166 00:23:32,650 --> 00:23:39,100 Then these sign ups is what we call a we'll call approach these sign ups and all. 167 00:23:39,100 --> 00:23:47,530 You can make these sign ups where exclusive contracts hail B and C and mechanical tactics I've seen since. 168 00:23:47,530 --> 00:23:54,100 And so you have to persuade you can do recognition this way or this way. 169 00:23:54,100 --> 00:24:01,830 But when you take exotically, that's the word from. A lot of work before arriving in the lab. 170 00:24:01,830 --> 00:24:08,800 What you should give them to decide what you can see that he was able to make some money at the junction. 171 00:24:08,800 --> 00:24:15,250 OK, and the junction you have just the exchange of the Chelsea strength here and with the energy, 172 00:24:15,250 --> 00:24:19,590 you get to this to ensure the Chevron junction, sorry. 173 00:24:19,590 --> 00:24:26,620 And but year much prefer to Theresa arbitrary detention than prefers to make it. 174 00:24:26,620 --> 00:24:34,580 And so. Because of this past way, we always hope that whatever you escape will do is OK. 175 00:24:34,580 --> 00:24:39,080 Just make excel, be active so that now we can finish the job. 176 00:24:39,080 --> 00:24:45,440 And in fact. Surprise, surprise. That was unexpected. 177 00:24:45,440 --> 00:24:52,370 In fact, the fastidious, activated but effective leads to the first sign of exchange. 178 00:24:52,370 --> 00:25:00,220 And so now you make a mixture of the charade junction and swords of. 179 00:25:00,220 --> 00:25:05,030 OK, that's the chromosome damage regulation best way. OK. 180 00:25:05,030 --> 00:25:09,770 Mobile element plasmids. Phage I makes. 181 00:25:09,770 --> 00:25:12,320 I mean, they want to use Excel, 182 00:25:12,320 --> 00:25:21,130 but they don't want to depend on if GSK because GSK makes you active only a tiny fraction of the sales cycle, that's a specific position. 183 00:25:21,130 --> 00:25:24,320 When there is said next to this television, Mashele, show me, 184 00:25:24,320 --> 00:25:32,470 many just want to preserve your dominance when you need it and ensure a fish you want to integrate when you until the say you don't, 185 00:25:32,470 --> 00:25:39,580 we don't want to wait. Which can take days in the environment two days before the events. 186 00:25:39,580 --> 00:25:46,710 And so that's. What the plasmid and the phages are using most of them. 187 00:25:46,710 --> 00:25:53,190 And so the plasmid, the involved, these two very nice, beautiful systems, 188 00:25:53,190 --> 00:26:01,080 so they have two core recommendation sites, but they are sea sequences next to this. 189 00:26:01,080 --> 00:26:13,790 So the of sequences which are going by sea factors which force the formation of proteins in China, which is protocol. 190 00:26:13,790 --> 00:26:19,400 And then he tells you he makes the quality dongsheng. 191 00:26:19,400 --> 00:26:22,670 This old gentleman can either be stable, stable, 192 00:26:22,670 --> 00:26:34,570 stabilised so that replication will from this water can recreate Truman or Mills and James Earl and the dying can be shipped back. 193 00:26:34,570 --> 00:26:47,200 Or. They are even less need that force door injunction, and then Ms Excel Rd do the second step and so you directly go to make a donation. 194 00:26:47,200 --> 00:26:55,240 So that's plasmids. So now I'm going to introduce you a few things about your career. 195 00:26:55,240 --> 00:27:00,010 Just to introduce to the integrated mobile elements, so they built your career. 196 00:27:00,010 --> 00:27:11,430 It's a bit like a teaching. Most friends of your career are non-pathogenic the environmental. 197 00:27:11,430 --> 00:27:16,050 There is only two two-syllable of the value Colella, which we can. 198 00:27:16,050 --> 00:27:18,960 We give rise to the pandemics. 199 00:27:18,960 --> 00:27:33,480 And um, and so and on top of that, they need to acquire the Kodaira toxin and the cholera toxin is born on a knife makes the cholera causing phage. 200 00:27:33,480 --> 00:27:41,610 And so these phage integrate into the site of the first chromosome and sometimes the second chromosome didn't tell you. 201 00:27:41,610 --> 00:27:44,070 Cuomo asked, Do you believe it was two chromosome? 202 00:27:44,070 --> 00:27:52,440 Which is kind of interesting because when you start thinking about only chromosome, you can have much more problems. 203 00:27:52,440 --> 00:28:00,390 And so I'm going to just to tell you how City integrates into the sites and I'm going to introduce you a second element, 204 00:28:00,390 --> 00:28:12,700 which is a toxin encrypted phage. So first, you know, we can see that, in fact, that only the creative team phage. 205 00:28:12,700 --> 00:28:16,500 Indeed, you have also the Chelsea phage. You can have virus one. 206 00:28:16,500 --> 00:28:25,050 You can have all the type of page of your favourites and you make arrays so you can integrate one and then the second one and then reintegrate. 207 00:28:25,050 --> 00:28:32,190 And so and so and that's because those elements of the phage. And so that's how it happens. 208 00:28:32,190 --> 00:28:40,060 You have the first Timex, which carries an upside and uses this as a B sides, and so it creates injuries, 209 00:28:40,060 --> 00:28:45,660 and now you have a metal side and then that's also swinging the two integrated element. 210 00:28:45,660 --> 00:28:51,240 The guitar is always the deep side. This is good for the Senate. 211 00:28:51,240 --> 00:28:59,870 This is good for the element, because if you don't free creative side, then you'll say is fix your minus and so it will be lost in the environment. 212 00:28:59,870 --> 00:29:03,050 And then you have the second element that can integrate. 213 00:29:03,050 --> 00:29:11,420 And so if you look at where is the Deep South and the series of the elements, you can see which one was the element integrated for. 214 00:29:11,420 --> 00:29:19,400 So in this case, I drew. She has integrated first and then Korea because that's what we observe in the evolution in the pathogenic strains. 215 00:29:19,400 --> 00:29:24,830 You always have see before you are. 216 00:29:24,830 --> 00:29:35,390 OK, so when we look at it, that's what it takes to have two beautiful eggshell recombination sites. 217 00:29:35,390 --> 00:29:43,400 The problem that the self-recrimination cites as a good model on the sea to the exchange was this. 218 00:29:43,400 --> 00:29:47,750 But it a wrong spacing fights. There's 12 nucleotide instead of six. 219 00:29:47,750 --> 00:29:57,740 So 12 is Alpha Alex term. So basically the two reconvene is instead of facing each other like that, they would be facing original like that. 220 00:29:57,740 --> 00:30:04,250 This is not possible to do anything that the second most as the perfect spacing you can buy. 221 00:30:04,250 --> 00:30:10,280 You can make connexion between the communities, but you have no meraji to do a recommendation. 222 00:30:10,280 --> 00:30:20,280 So that was a problem. And the problem was solved because, in fact, the genome of the phage single stranded DNA in on the genome of the phage, 223 00:30:20,280 --> 00:30:27,810 those two elements are in opposite orientation. And so they form the stem of a hairpin. 224 00:30:27,810 --> 00:30:34,350 And then this time of wrapping, thanks to the second spacing, you have the good spacing. 225 00:30:34,350 --> 00:30:39,150 And here you have the good energy. And so he can do the catalyses. 226 00:30:39,150 --> 00:30:46,770 And so it can catalyse this wheel. Sites with defend and you get into A. You can get. 227 00:30:46,770 --> 00:30:59,040 You can enter into it. The second element I'm going to introduce is to chance the toxin encrypted, which is always integrated, defrosted. 228 00:30:59,040 --> 00:31:09,180 So this element as a perfect spacing, it has a perfect central region, two or two steps of communication. 229 00:31:09,180 --> 00:31:14,080 And so this element uses the device with. OK. 230 00:31:14,080 --> 00:31:19,500 To get. What are you doing made by day and you get 20 attention? 231 00:31:19,500 --> 00:31:28,050 There's a ritual made by Cairo. You don't you don't want to depend on the state. 232 00:31:28,050 --> 00:31:34,900 And so this element. Eyeballs its own Xia activation factor. 233 00:31:34,900 --> 00:31:40,180 OK. It's a different type of factor that can activate Excel. 234 00:31:40,180 --> 00:31:46,030 If this gets a different food, it's a different semi. That's OK. 235 00:31:46,030 --> 00:31:52,450 Now there is a problem with this, which I do not introduce. You just kept it fast. 236 00:31:52,450 --> 00:32:00,580 If you look at the type of TLC and you look at the second flight, so that's not an easy one sight. 237 00:32:00,580 --> 00:32:06,250 If you put all these elements, you can see accepts the binding you predict. 238 00:32:06,250 --> 00:32:09,860 You don't see binding. You put the two of them together. 239 00:32:09,860 --> 00:32:18,130 You don't see any excess. Shall we find that the comparison with when you produce, you get the two C and D bone? 240 00:32:18,130 --> 00:32:26,070 So you have. So this site doesn't make excel, you cannot buy, so you cannot make a sign ups. 241 00:32:26,070 --> 00:32:32,170 You have just one. And so just for some truths kind of intrigued us. 242 00:32:32,170 --> 00:32:38,940 So we wanted to compare the mechanism of activation of a system on this type of site. 243 00:32:38,940 --> 00:32:45,540 And we said, Oh, we are not just going to sell this site, we are just going to study all the possible sites. 244 00:32:45,540 --> 00:32:51,510 Yes. So we took a frontal attack on sites and we just tested them. 245 00:32:51,510 --> 00:32:56,340 So this is just this integrated task. 246 00:32:56,340 --> 00:33:03,190 Exactly. Chelsea cannot be integrated, if you say, but if we take those elements, we can get. 247 00:33:03,190 --> 00:33:13,930 Some of them can be integrated. Twenty five percent of them, if we use exact now, we have definitive rates as well. 248 00:33:13,930 --> 00:33:18,040 As well at the key from issue now is very, 249 00:33:18,040 --> 00:33:26,320 very intuitive and those elements now that was 2.5 years and now twenty five percent of the sequence can be integrated. 250 00:33:26,320 --> 00:33:31,390 So he just showed you. If we look into the detail of the sequence. 251 00:33:31,390 --> 00:33:38,680 So this is a map where I'm showing the city side and you do the sequence as one, two, 252 00:33:38,680 --> 00:33:43,720 three or four different scenes from the episode, I'm just showing them on the left right. 253 00:33:43,720 --> 00:33:54,250 And and so those are the sequence which have one difference to three or four to difference from the episode, but which are close up to this. 254 00:33:54,250 --> 00:34:04,980 And so I think make them feel sick and make them recombine and make them integrate, but still many of them are very badly integrated. 255 00:34:04,980 --> 00:34:10,920 And once you put feel you can make almost insect free online. 256 00:34:10,920 --> 00:34:16,410 Whether it's closer or closer to two to nothing. 257 00:34:16,410 --> 00:34:28,770 OK, so 15 makes the possibility to recombine again sites which have almost no more to do with a true site where we can buy. 258 00:34:28,770 --> 00:34:31,230 So that's just the last thing. 259 00:34:31,230 --> 00:34:40,570 So what I wanted to say about Excel is that it's a highly versatile communication machinery because you can do interval of mechanistic, 260 00:34:40,570 --> 00:34:50,260 you have similar pathway, you can do the best way. Chromosome demoralisation by its kids, you'd see exactly, 261 00:34:50,260 --> 00:34:58,420 but you can also do the sequence where you can stop at the replication stage with Citrix or Cody one, or you can do the 40 plus way. 262 00:34:58,420 --> 00:35:03,870 So in terms of mechanistic, you can do many, many things. It's completely. 263 00:35:03,870 --> 00:35:10,230 And if you look at the sites you can act on. In fact, we can on sites which are very, very well. 264 00:35:10,230 --> 00:35:16,860 So here I just told you that you can put whatever you want here, basically, and it will be able to recombine. 265 00:35:16,860 --> 00:35:25,890 So I told you before that, it is a very stringent, highly regulated, but in fact, you can make it do almost anything you want. 266 00:35:25,890 --> 00:35:30,510 You can make do well. And the only way, I mean. 267 00:35:30,510 --> 00:35:38,040 Maybe I'm wrong, but I think the only type of virus in communities which I know that we're all 268 00:35:38,040 --> 00:35:46,030 able to produce so versatile in some of the sites of the integrase and so and. 269 00:35:46,030 --> 00:35:51,110 If you look in the genetic tree of. So that's three of them. 270 00:35:51,110 --> 00:36:01,420 That's the exterior recommendations. They still deal very, very closely related and so narrow and. 271 00:36:01,420 --> 00:36:09,550 But they are close to the integrase mean the thousand of the communist from sealed people's integrations. 272 00:36:09,550 --> 00:36:17,260 So I just wanted to say that I think we can speculate again in the initially. 273 00:36:17,260 --> 00:36:22,030 A long time ago, there was this type of family of power in their communities, 274 00:36:22,030 --> 00:36:30,970 and they've been used on one side for the integrase where the integral is just recovered 275 00:36:30,970 --> 00:36:37,420 and this silly domain which allowed them to work on those sites and on the other side. 276 00:36:37,420 --> 00:36:43,000 These these three communities evolve to be stringently regulated. 277 00:36:43,000 --> 00:36:48,040 And so one first thing to help regulators to. 278 00:36:48,040 --> 00:36:53,590 Separate into two communities to force the fact of using two different sites, 279 00:36:53,590 --> 00:37:02,920 and so I would say that building was your, you know, one and evolved to block it from acting all the time. 280 00:37:02,920 --> 00:37:08,160 And then on top of that, the regulation is key. 281 00:37:08,160 --> 00:37:15,220 Yeah. So that's just to say that's a right and they sure etc. in the same meeting. 282 00:37:15,220 --> 00:37:27,370 Yeah. So just as a switch, that's the people I enjoy having in the lab or me are a little after. 283 00:37:27,370 --> 00:37:36,430 And also, I had some on top of gen. I had the chance to be sitting next to John Rawls doing a sabbatical and then to each other. 284 00:37:36,430 --> 00:37:39,790 And then I met thousands from place, I'm sure. 285 00:37:39,790 --> 00:37:58,520 Well. And that's the people that are following the trace of Dave trying to talk you more and more, the things were false theories. 286 00:37:58,520 --> 00:38:03,380 If your names are not at the right place because some people have work at different places to say, 287 00:38:03,380 --> 00:38:09,010 you can work on these loans, you just on this one loan this one. 288 00:38:09,010 --> 00:38:22,070 OK, thank you. Thank you. Also, we have time for one question. 289 00:38:22,070 --> 00:38:31,140 Then. So do you know anything about how safety activates sexyold? 290 00:38:31,140 --> 00:38:42,180 Know how it activates excelled, you know what we just know that's exactly on top of activating the Sylvie's making it forming a sign ups. 291 00:38:42,180 --> 00:38:48,090 But the mechanism? We don't know it interacts directly with, but we don't know. 292 00:38:48,090 --> 00:38:53,710 I mean, even if I'm not sure we know it's activated. 293 00:38:53,710 --> 00:38:55,320 But that's a that's a good question. 294 00:38:55,320 --> 00:39:11,580 We hope that discussing with people will get at some points of chromium, which you may need to discuss with Yemen, but that's not the question. 295 00:39:11,580 --> 00:39:15,090 So Vibrio cholerae has two chromosomes, doesn't it? 296 00:39:15,090 --> 00:39:23,720 We have the the earlier telecon about two. And so one of the chromosomes is very accessible to transposition and exchange rate is more stable. 297 00:39:23,720 --> 00:39:29,370 Could these systems be operating designed to to tweak between the two chromosomes, do you think? 298 00:39:29,370 --> 00:39:37,610 Or is that something? In one of chromosome favouring the transposon type integration, the other one or is that not mono? 299 00:39:37,610 --> 00:39:42,320 In fact. I mean, all the variables are to chromosome. 300 00:39:42,320 --> 00:39:49,790 So one is a true chromosome. It's a general chromosome. The second one is the plasmid, which is inside. 301 00:39:49,790 --> 00:39:56,420 So they are called comets because they are important. 302 00:39:56,420 --> 00:40:02,990 And in the videos, so you have the elements on the two chromosome, but in them for your career, for example, 303 00:40:02,990 --> 00:40:10,130 the to be pathogenic, you need the toxin called the ratepayers to a deal and then decide on the Fox chromosome. 304 00:40:10,130 --> 00:40:18,710 It's a big genetic island. And uh, but on the second chromosome, you have a super zero. 305 00:40:18,710 --> 00:40:23,190 But in other species, you have the shift on different forms of sex chromosomes. 306 00:40:23,190 --> 00:40:28,040 So it's not the reason why you have two chromosomes. 307 00:40:28,040 --> 00:40:38,200 Not. Not just to I mean, nobody know why some people have speculated that because you have to chromosome you truly genes, 308 00:40:38,200 --> 00:40:43,270 you can divide faster and the values are very fast. 309 00:40:43,270 --> 00:40:48,730 So maybe you can divide every 10 minutes, but that doesn't fit freely with all the mothers, 310 00:40:48,730 --> 00:40:55,090 so they'll know at the moment there is no good reason to say why they have a second chromosome. 311 00:40:55,090 --> 00:41:03,610 OK, thank you. So the next picture of this afternoon is so Rodrigo Reis llama. 312 00:41:03,610 --> 00:41:10,030 It could come today, so we will have Rodrigo by the bone zoom. 313 00:41:10,030 --> 00:41:20,170 And Rodrigo, this is with David, I think, early 2000 year 2000 and stay here for a postdoc. 314 00:41:20,170 --> 00:41:25,720 And at the end of that, he got a position at McGill University. 315 00:41:25,720 --> 00:41:29,230 And this is where he is and is. 316 00:41:29,230 --> 00:41:34,570 I hope he's going to join us out of that. 317 00:41:34,570 --> 00:41:42,430 Yeah, yeah. So going to talk about replication? 318 00:41:42,430 --> 00:41:50,200 I did it. Yeah, hi. 319 00:41:50,200 --> 00:41:53,860 Yeah, I did change my title. Sorry about that. 320 00:41:53,860 --> 00:41:59,470 I thought it was more appropriate. Forty four for what it was. 321 00:41:59,470 --> 00:42:13,660 What I want to talk about. And well, first, I'm assuming that you can listen to me and that you are looking at the slides. 322 00:42:13,660 --> 00:42:29,740 So I'll just continue. So I first have to thank Lydia and the rest of the people that helped organising the event to invite invite me. 323 00:42:29,740 --> 00:42:37,150 I'm happy to participate in the symposium celebrating Dave and uncertain, 324 00:42:37,150 --> 00:42:44,470 missing all the lunches and dinners and all the good company of the people that are there. 325 00:42:44,470 --> 00:42:55,150 [INAUDIBLE] have to be next time. OK. 326 00:42:55,150 --> 00:43:03,430 Yes, one second. All right. So what I'm I'm going to be telling you about today. 327 00:43:03,430 --> 00:43:11,740 It's a recurring question that I have in my work, which is how during DNA replication, 328 00:43:11,740 --> 00:43:22,540 which implies the separation of the two strands and the synthesis of these of the of two new complementary strands. 329 00:43:22,540 --> 00:43:32,240 How is that the cell managed to prevent the the excessive accumulation of single strand DNA? 330 00:43:32,240 --> 00:43:43,480 This is an important question because single strand DNA is known to be more prone to DNA breaks and has a higher 331 00:43:43,480 --> 00:43:52,540 tendency to accumulate based modifications like domination and therefore leads to a higher mutation rate. 332 00:43:52,540 --> 00:44:07,960 So one aspect that we study in terms of trying to answer understand this question is looking at the dynamics and the architecture of the replication. 333 00:44:07,960 --> 00:44:18,340 And so what I'm going to be telling you about is the these these aspects of the of the eukaryotic replication. 334 00:44:18,340 --> 00:44:25,990 But before starting my scientific the book, 335 00:44:25,990 --> 00:44:36,130 I would like to go back several years and tell you my personal perspective of what 336 00:44:36,130 --> 00:44:44,980 was going on in day five and how when I when I got there and how that has influence, 337 00:44:44,980 --> 00:44:52,030 what what I've done in India in the rest of my career. 338 00:44:52,030 --> 00:44:58,000 So I got there in 2005, and that was a really exciting time, I think. 339 00:44:58,000 --> 00:45:02,560 And Bernard already mentioned a little bit about that. 340 00:45:02,560 --> 00:45:10,570 So at the time, Sinden and Christof had recently published a paper in Gene Sundeep, 341 00:45:10,570 --> 00:45:23,410 where they really started describing how their nuclear ecology it's organised into, like, is really it. 342 00:45:23,410 --> 00:45:30,190 It contains an organisation that is inherited across many generations, and so it's very stable. 343 00:45:30,190 --> 00:45:39,760 So you have the origin in the middle and and the replicators are decides on a lot of questions so that that was completely surprising. 344 00:45:39,760 --> 00:45:47,800 And then they they delve into they went into trying to understand how those was this changing during the cell cycle and so on. 345 00:45:47,800 --> 00:45:53,860 And there were many questions that this opened many questions, some of them. 346 00:45:53,860 --> 00:46:00,550 Another surprising observation was one year after these first publication. 347 00:46:00,550 --> 00:46:12,400 Now, with Sean, where they observed that there was also a particular arrangement between the two sister chromosomes. 348 00:46:12,400 --> 00:46:17,320 So there were many questions that were opened amongst them. 349 00:46:17,320 --> 00:46:21,980 But I find probably most important were what? What is that? 350 00:46:21,980 --> 00:46:29,740 Is structure in this nucleus? What are the components of the process to study our structure in this nucleus? 351 00:46:29,740 --> 00:46:39,790 And how is that DNA segregation is happening? Some of it I was able to participate in answering the question. 352 00:46:39,790 --> 00:46:53,770 Part of the question of segregation with seemed Wang a few years after where we describe the function of the perfor in the process. 353 00:46:53,770 --> 00:47:09,730 Let me see. And then I was also quite involved in studying the role of Mok Be the assembly complex Macbeth, 354 00:47:09,730 --> 00:47:20,740 which was something that was initiated by Christoph and Alicia and was first published in this paper in 2008, 355 00:47:20,740 --> 00:47:28,630 where the word to describe the effect of in the organisation of the chromosome and the effect of losing Macbeth. 356 00:47:28,630 --> 00:47:43,150 And this project was followed on by a few other people that I interacted closely with Marina and being on and 357 00:47:43,150 --> 00:47:53,330 later taken on by angina that that's going to talk after me where she culminating in the eye in two papers. 358 00:47:53,330 --> 00:48:06,490 One. Where we described how the just the expression of mock beef cooked without any DNA replication could restructure the chromosome, 359 00:48:06,490 --> 00:48:19,000 and in a second quarter, we looked at in a second paper where we looked at the structure of memory, remarkably young Stefan. 360 00:48:19,000 --> 00:48:23,260 So in my time, these were people that I interacted a lot with. 361 00:48:23,260 --> 00:48:27,580 But there were many order of wonderful people that generated this amazing, 362 00:48:27,580 --> 00:48:39,340 stimulating environment that I think I have everyone in here that was in indeed life I love at the time. 363 00:48:39,340 --> 00:48:47,050 I I, I guess I excluded a part two students that were but graduates and both Dr. and 364 00:48:47,050 --> 00:48:56,390 and some recurring members such as Marcelo that could come every summer are here. 365 00:48:56,390 --> 00:49:03,190 And so this this was like really a fantastic environment for for ideas and to start working. 366 00:49:03,190 --> 00:49:09,640 And we did have fun. And I have here two examples. 367 00:49:09,640 --> 00:49:22,510 One, the first one is in 2008. This was the first of all UBC tie dinner where you can see they are looking very sharp. 368 00:49:22,510 --> 00:49:35,560 And the second is in one of our Christmas lunch at the anchor, but we went a few times that the same buffet, it was really fun and on. 369 00:49:35,560 --> 00:49:42,130 But in this in this occupation, we all wear these wicked red week. 370 00:49:42,130 --> 00:49:55,270 And I think we all look fabulous. But I want to draw your attention to the style of which Dave wears the week. 371 00:49:55,270 --> 00:50:01,960 I think it's fantastic. Okay. 372 00:50:01,960 --> 00:50:10,240 So it was in this environment with this fantastic group of people that I started trying to 373 00:50:10,240 --> 00:50:17,050 contribute to the understanding of of the of the chromosome structuring and segregation. 374 00:50:17,050 --> 00:50:21,160 And and I chose to focus on the end of replication. 375 00:50:21,160 --> 00:50:35,230 And the idea of these was that I wanted to test whether DNA replication was contributing to segregating the chromosomal loci. 376 00:50:35,230 --> 00:50:45,100 To do this, I studied first studied the the position and the location at which DNA replication was occurring in the cells. 377 00:50:45,100 --> 00:50:57,850 But later on, with Dave's support and encouragement, I started branching out, leaving the chromosome. 378 00:50:57,850 --> 00:51:07,780 The study of the chromosome structure on a side and focussing more on the fundamental aspects of DNA replication. 379 00:51:07,780 --> 00:51:12,400 And this led me to a collaboration of wonderful collaboration with Martinique, 380 00:51:12,400 --> 00:51:21,850 where we described the stoichiometry of the reptilian observing that one of us has three polymerases 381 00:51:21,850 --> 00:51:29,830 or some single molecule studies also included Stephane and I mentioned the MOBI paper with Anja, 382 00:51:29,830 --> 00:51:38,680 and Stefan was an author. And then more later on in my life, 383 00:51:38,680 --> 00:51:53,260 I continued studying aspects of DNA replication and how this has led to the description of the bacterial replication as a very dynamic complex 384 00:51:53,260 --> 00:52:06,760 where the DNA polymerase on order most basically most of the subunits in the reptilian are exchanging are being replaced every few seconds. 385 00:52:06,760 --> 00:52:12,610 We're still trying to understand what what is the biological relevance of this, 386 00:52:12,610 --> 00:52:26,050 but likely has to do with a more efficient the DNA repair and and maybe dealing with excessive super coiling. 387 00:52:26,050 --> 00:52:50,160 So. So then we it's following this same type of questions and ideas, I now try applying this to the less understood repetition in in eukaryotic cells. 388 00:52:50,160 --> 00:53:01,280 So I have the idea of these was to try developing single molecule microscopy to basically, 389 00:53:01,280 --> 00:53:09,970 as I mentioned, try answering like similar questions in this in this system. 390 00:53:09,970 --> 00:53:24,400 And once I'm going back to trying to understand how is that the rep also manages to coordinate the the two activities, 391 00:53:24,400 --> 00:53:29,350 the Healy case and the various sentences and the are the Ruptly. 392 00:53:29,350 --> 00:53:40,270 Some in eukaryotes is particularly interesting because there there is no known 393 00:53:40,270 --> 00:53:46,270 interaction between the large instant polymerase pulled Delta and the helix, 394 00:53:46,270 --> 00:53:52,360 this EMG helix. Hence, this suggests a model were pulled out. 395 00:53:52,360 --> 00:54:01,120 A different copy of Pearl Delta is required every five about every five seconds, every Okazaki fragment. 396 00:54:01,120 --> 00:54:06,280 So, and furthermore, it suggests that for four days, 397 00:54:06,280 --> 00:54:14,390 coordination between DNA synthesis and unwinding to work there has to be like an efficient turnover pulled up. 398 00:54:14,390 --> 00:54:23,560 We would try to test this model and also try to trying to understand older aspects of the eukaryotic replication. 399 00:54:23,560 --> 00:54:35,830 We went on, as I mentioned, to to set up a single molecule microscopy in budding yeast. 400 00:54:35,830 --> 00:54:46,060 This is a summary of what of our particular experimental procedures. 401 00:54:46,060 --> 00:54:50,470 We use scale attack fusions of different reptiles. 402 00:54:50,470 --> 00:54:57,550 Some surviennent we use. We chose scale attack because this allows us to utilise organic flow of force, 403 00:54:57,550 --> 00:55:05,440 which are superior in intensity and and four to stability to fluorescent Brookings. 404 00:55:05,440 --> 00:55:09,880 Then we use an old trick in microscopy, 405 00:55:09,880 --> 00:55:15,910 which is using integration times of 500 milliseconds when looking at single molecules 406 00:55:15,910 --> 00:55:22,630 so that if a molecule is bound to chromatin is not going to be moving that much. 407 00:55:22,630 --> 00:55:26,320 Therefore, we are going to be able to observe them as spots. 408 00:55:26,320 --> 00:55:34,640 But if they are diffusing, then we can see them like all over the place or blurring the intensity they they must. 409 00:55:34,640 --> 00:55:37,690 And so we don't we don't detect them as spots. 410 00:55:37,690 --> 00:55:45,780 We use High-Low or Applicator to increase the signal to noise, and then we use a single two activation events. 411 00:55:45,780 --> 00:55:49,660 So this this later this day that we are using is for Turkey. 412 00:55:49,660 --> 00:55:58,180 Available is coming from the Gemili Farms. So we use a single for two activation event and then we watch over time. 413 00:55:58,180 --> 00:56:02,740 And what we are recording try to figure out. And here it is. 414 00:56:02,740 --> 00:56:10,480 What is the lifetime of these spots? Since the lifetime of this, but once we correct for photo bleaching, 415 00:56:10,480 --> 00:56:18,820 give salts the an estimate of the dwell time of this protein, some chromatin planning the replacement. 416 00:56:18,820 --> 00:56:26,470 So these are examples. This is this is histone H3. 417 00:56:26,470 --> 00:56:35,480 [INAUDIBLE] attack using the protective variable J if i 04:09 that we got from the farms reaffirms the strain. 418 00:56:35,480 --> 00:56:41,380 Mixing also comes from a Tatiana capo was left. 419 00:56:41,380 --> 00:56:45,430 And so you can clearly see most. In this case, it's Keystone. 420 00:56:45,430 --> 00:56:50,020 So most of the proteins are going to be bound to chromatin. You can see a lot of spots. 421 00:56:50,020 --> 00:57:04,900 Occasionally, you can see that there's things moving blur the spots that are of molecules moving very fast. 422 00:57:04,900 --> 00:57:15,100 We know that we are looking at single copies of Ruptly some subunits because the spots that we observe, they they look like single molecules. 423 00:57:15,100 --> 00:57:21,460 They move like single molecule, and they also are sensitive to stress. 424 00:57:21,460 --> 00:57:32,770 And what I mean is that when we look at those spots, we find examples of the spots bleaching in a single step, 425 00:57:32,770 --> 00:57:39,130 which is a golden sand in the single molecule field. 426 00:57:39,130 --> 00:57:46,360 But also, if we look at the distribution of the intensity of these spots, we see unique animal distribution. 427 00:57:46,360 --> 00:57:56,530 Once more are suspected for single molecules. Then when we look at their movement, particularly at the diffusion coefficient on the alpha coefficient, 428 00:57:56,530 --> 00:58:07,640 the anomalous coefficient of these spots, it fits with what has been previously described for chromosomal loci. 429 00:58:07,640 --> 00:58:19,290 You're seeing. The lack operator of race previously in East, finally, if we treat cells with hydroxyurea, 430 00:58:19,290 --> 00:58:30,120 we can observe that the dwell time of the single strand buying protein RPA goes from 19 sorry, from nine to 18 seconds. 431 00:58:30,120 --> 00:58:45,240 Therefore, of reinforcing the idea that nut cells are the replication forks are being sold and cells are the RPA staying longer in there. 432 00:58:45,240 --> 00:58:49,620 So cells are really active in replication. 433 00:58:49,620 --> 00:58:55,170 The cells that we are looking at are really active in replication and and and all this gives us 434 00:58:55,170 --> 00:59:03,840 confidence that we are really looking at the proteins that are participating in direct replication. 435 00:59:03,840 --> 00:59:12,930 A few a couple more things since we wanted to be able to look at 12 times that span from a few seconds to a few minutes, 436 00:59:12,930 --> 00:59:19,680 we we use different intervals between pictures. One second, a second in 20 seconds. 437 00:59:19,680 --> 00:59:27,450 So if we if we use a one second interval, we we just looked at a single imaging plane. 438 00:59:27,450 --> 00:59:32,100 But when we use these are longer range problems. 439 00:59:32,100 --> 00:59:40,110 We we included three imaging planes to minimise the possibility that when we were seeing the disappearance of a spot, 440 00:59:40,110 --> 00:59:51,630 it was because it was getting out of focus. Finally, we also use a PC and a neon green to determine which were the cells that were undergoing space. 441 00:59:51,630 --> 00:59:58,650 So we segment that the nuclei of those face using this label. 442 00:59:58,650 --> 01:00:01,860 From all these type of experiments. 443 01:00:01,860 --> 01:00:13,200 We generated this type of plots where we are looking at the lifetime, the distribution of lifetime of this, both for a for a particular time. 444 01:00:13,200 --> 01:00:22,890 So we need, you know, like in a particular interval, we feed it to a single exponential function and we corrected for bleach for each time. 445 01:00:22,890 --> 01:00:33,810 And from here, we as I mentioned, we generated we obtained the an estimate of the dwell times and we did this for a lot of 446 01:00:33,810 --> 01:00:40,860 different subunits covering the different parts of the reptiles on the CMG helicase, 447 01:00:40,860 --> 01:00:50,040 different polymerases and other components of the reptilian core components of the represen using different intervals. 448 01:00:50,040 --> 01:01:01,170 This is the summary of what we obtained and so reassuringly expectedly, 449 01:01:01,170 --> 01:01:11,760 but reassuringly still, we observed that not the CMG helicase and polyethylene, 450 01:01:11,760 --> 01:01:16,590 which were expected to bind to chromatin for relatively long time, 451 01:01:16,590 --> 01:01:28,200 like several minutes at least, where indeed we found them to be binding for four months. 452 01:01:28,200 --> 01:01:38,490 We also observed again reassuringly, that RPA was in contrast, binding for few seconds. 453 01:01:38,490 --> 01:01:50,680 I suspect it for for the single strand DNA binding protein, which is replaced actively during synthesis of Okazaki fragments. 454 01:01:50,680 --> 01:01:57,010 Now, there were few surprising observations in our data. 455 01:01:57,010 --> 01:02:10,570 The first is that full alpha, which interacts with this stable Syrian city for what we thought is known to interact with City four, 456 01:02:10,570 --> 01:02:25,390 was bound, therefore only 60 seconds. We were expecting it to be stable part of the complex and and it's it has an intermediate and dwell time. 457 01:02:25,390 --> 01:02:33,840 This means that a single copy of Pool Alpha can synthesise few parameters. 458 01:02:33,840 --> 01:02:37,290 Before I refer refer, 459 01:02:37,290 --> 01:02:49,950 before we went forward and try breaking this interaction with CTF four and we use this mutant point mutation that that doesn't bind anymore to 384. 460 01:02:49,950 --> 01:03:03,570 And surprisingly, we also serve that we can still detect relatively long and binding of 40 seconds and not pull off up to Cromarty. 461 01:03:03,570 --> 01:03:10,110 We still this is since we are still in the descriptive part of the of this part of the study, 462 01:03:10,110 --> 01:03:20,020 so we are trying to understand quite what is what is this telling us? 463 01:03:20,020 --> 01:03:26,440 But by far, the most surprising thing was that we observe that pulled out of the lagging strand polymerase 464 01:03:26,440 --> 01:03:32,650 that I was telling you that we were expecting it to be replaced every few seconds, 465 01:03:32,650 --> 01:03:40,750 we observe it to be bound for about five minutes. 466 01:03:40,750 --> 01:03:48,580 So there's a few possible interpretations of these of this long binding upon Delta. 467 01:03:48,580 --> 01:03:55,090 The first is that maybe what we are observing is that the pull there's up, 468 01:03:55,090 --> 01:04:03,460 there's copies of Pearl Delta that are acting out the larkin's drum and the lines drawn and that are replaced every few seconds. 469 01:04:03,460 --> 01:04:11,260 But we have orders that bind somewhere else, maybe on the leading strand or poorer place. 470 01:04:11,260 --> 01:04:17,510 It doesn't really matter. But that that could last for a much longer time. 471 01:04:17,510 --> 01:04:23,050 And that's. OK. 472 01:04:23,050 --> 01:04:31,090 When I ask him, fine. OK, so sorry. 473 01:04:31,090 --> 01:04:40,900 OK, so I'll I sorry, I didn't keep track of the time. 474 01:04:40,900 --> 01:04:51,410 Well, so I'll just I'll just say that. 475 01:04:51,410 --> 01:04:59,530 I'll just mention that that we think that pulled out is interacting directly to the the rest of the reports. 476 01:04:59,530 --> 01:05:04,030 And and that this suggests that other Saudis are trombone model also for 477 01:05:04,030 --> 01:05:09,100 eukaryotic and has implications for fragment maturation or primitive inheritance. 478 01:05:09,100 --> 01:05:16,090 And and we are trying to understand what, what, order, what, what, 479 01:05:16,090 --> 01:05:28,300 what is the biological function of all these and and what other proteins that interact with Iraqis on how how is that they are interacting? 480 01:05:28,300 --> 01:05:41,140 And so. So I mean these these are the people that did the work, particularly knitting Kapadia and and the funding agencies. 481 01:05:41,140 --> 01:05:45,970 And we have an open position for postdocs to continue work this work. 482 01:05:45,970 --> 01:05:55,630 And I just want to finish with thanking Dave again for all his support and encouragement over the years. 483 01:05:55,630 --> 01:06:01,960 And this is another nice picture of him in a black tie. 484 01:06:01,960 --> 01:06:13,160 Thanks. Sorry for going over time. Thank you. 485 01:06:13,160 --> 01:06:17,840 Thank you, Rodrigo. You do you help believe me? Yes. 486 01:06:17,840 --> 01:06:22,370 OK, fine. So thank you for this nice presentation. 487 01:06:22,370 --> 01:06:34,530 I think we are running a little bit out of time, but maybe, maybe there is space for one quick question. 488 01:06:34,530 --> 01:06:42,580 Look. So maybe I had one Rodrigo's still there, though, it's gone. 489 01:06:42,580 --> 01:06:58,030 Yes. OK, so so is it no possibility to have multiple pull delta polymerase on the repeal zone with the data that you've shown? 490 01:06:58,030 --> 01:07:00,970 So no, we don't think so. 491 01:07:00,970 --> 01:07:11,500 And this is because one part of the the things that I didn't have time to to show you is that we we also try estimating what what is the fracture? 492 01:07:11,500 --> 01:07:17,020 What is the copy number of copies of Pearl Delta bound chromatin? 493 01:07:17,020 --> 01:07:27,550 And we obtain a number of a few hundred copies, which is just too close to the number of the replicas of the active reptiles something in the cell? 494 01:07:27,550 --> 01:07:32,290 Yeah. So I think I think our data is suggesting that it's not. 495 01:07:32,290 --> 01:07:37,210 Yeah. Thank you, Rodrigo. We hope to see you soon. 496 01:07:37,210 --> 01:07:42,440 Yeah. Here in Europe, and we wish you the best in my gill. 497 01:07:42,440 --> 01:07:52,440 So the next speaker is going to be Anjana. Yeah. 498 01:07:52,440 --> 01:07:56,880 So is John and Jenna around? Yeah, I'm here. 499 01:07:56,880 --> 01:08:07,600 Online from that. High, Anjana. So I, as I think the the the. 500 01:08:07,600 --> 01:08:18,850 The trajectory of angina is similar to that, the one of Rodrigo, so again, angina or disease with Dave just after, I think we go. 501 01:08:18,850 --> 01:08:22,390 And then she went on, but I don't remember where it was in the states. 502 01:08:22,390 --> 01:08:32,170 I can't remember where. And then she got to a position at the National Centre of Biological Science in Bangalore in India. 503 01:08:32,170 --> 01:08:44,060 And this is from where she's going to talk to us about DNA repair and searching for homology in during homologous recombination. 504 01:08:44,060 --> 01:08:52,750 OK, great. I'm going to quickly share my screen, make sure it's all OK. 505 01:08:52,750 --> 01:08:58,620 Can everyone see my screen? I can't hear you. 506 01:08:58,620 --> 01:09:06,890 OK. OK. Let me dive right in, so first of all, I am so, so sorry, I can't be there in person. 507 01:09:06,890 --> 01:09:14,330 Maybe there will be a time when these are not so difficult to apply for and then we will move around the world much easier. 508 01:09:14,330 --> 01:09:21,140 But for now, I am also really, really glad to be part of this really special day to celebrate this legacy. 509 01:09:21,140 --> 01:09:24,920 And so thank you, Lydia and everyone for having this online offline format. 510 01:09:24,920 --> 01:09:27,980 So many of us can be part of the city. 511 01:09:27,980 --> 01:09:38,450 So I am, as Bernard said, I'm on Janelle, but in writing and I started my lab at NGQDs in Bangalore in December 2016, 512 01:09:38,450 --> 01:09:49,130 and I was in the official lab between 2008 and 2012 as a graduate student that I studied beef and equal chromosome organisation. 513 01:09:49,130 --> 01:09:58,790 And so this is a drawing of an early B B dimer of Dimer that they've had rendered on his favourite black and red notebook. 514 01:09:58,790 --> 01:10:01,100 I'm pretty certain it's still there with him right now. 515 01:10:01,100 --> 01:10:08,990 He must carry one every time he goes to a seminar, either it's on him or under this chair, but I'm pretty certain he's building one so. 516 01:10:08,990 --> 01:10:20,420 And so what I want to do today is talk to you about a story from my own lab looking at the mechanism of bacterial homology, such during recombination. 517 01:10:20,420 --> 01:10:25,220 And the reason why I thought I'd talk about the story is for two reasons. 518 01:10:25,220 --> 01:10:33,470 One is because I think this for me from my side. I think it's truly a homage to Dave because I think our entire approach of trying to 519 01:10:33,470 --> 01:10:37,820 address this question comes from what I have learnt from him and being in his lab. 520 01:10:37,820 --> 01:10:44,360 And so you'll see a lot of signature of Dave in this looking inside cells doing in vivo biochemistry. 521 01:10:44,360 --> 01:10:52,310 Another reason why I thought this was particularly special for me to discuss is because it brings together two parts of science that I think 522 01:10:52,310 --> 01:10:59,990 Dave is super passionate about looking at the combination and also looking at structural maintenance of chromosome family of proteins. 523 01:10:59,990 --> 01:11:03,530 And in this story, both of them end up overlapping together. 524 01:11:03,530 --> 01:11:07,460 So I'm particularly excited to share this with you. Right. 525 01:11:07,460 --> 01:11:09,050 So very quickly. 526 01:11:09,050 --> 01:11:17,480 So in my lab, we are interested broadly in looking at DNA damage response and repair mechanisms to study how these impact genome maintenance, 527 01:11:17,480 --> 01:11:27,470 evolution, neurogenesis and survival. And we've had some beautiful introductions into double strand breaks apart from from several speakers already. 528 01:11:27,470 --> 01:11:33,560 And I'm going to just sort of summarise some key parts so that we can focus on the aspect that I'm interested in talking to you about today. 529 01:11:33,560 --> 01:11:38,330 So this will be able repairing a double strand because by homologous recombination, 530 01:11:38,330 --> 01:11:45,320 using an uncut copy of its DNA as a template for repairs and as illustrated by Steve Olarinde, 531 01:11:45,320 --> 01:11:49,190 et cetera, these are the early steps of recombination are really well characterised. 532 01:11:49,190 --> 01:11:56,960 We know that you need to reset both ends of DNA to reveal a single strand of DNA, and then that single strand of DNA is used for strand invasion. 533 01:11:56,960 --> 01:12:02,960 And then following that recombination and we already heard about the holiday junction. 534 01:12:02,960 --> 01:12:07,820 To understand this part of the combination, and we know that a key protein involved in this process, 535 01:12:07,820 --> 01:12:16,400 both for homology search as well as ground invasion, is a protein called recce in bacteria and its eukaryotic equivalent at 51. 536 01:12:16,400 --> 01:12:24,200 And so seminal work over several years now has established that it is absolutely essential for for recombination. 537 01:12:24,200 --> 01:12:29,810 We know that recce forms a nuclear protein filament on single strand of DNA that's revealed around the Blake site. 538 01:12:29,810 --> 01:12:35,060 But what we don't quite understand as yet is how the spreading nuclear protein filament orchestrates 539 01:12:35,060 --> 01:12:42,440 homology search to enable recombination and then illustrate why this is still an open problem in the field. 540 01:12:42,440 --> 01:12:47,840 So a lot of beautiful in vitro work establish that there are ways in which nuclear 541 01:12:47,840 --> 01:12:53,330 protein filament can move on DNA while engaging in one day sliding into segment hopping, 542 01:12:53,330 --> 01:13:01,910 all while engaging in microbiology sampling, looking for a homologous template, and once it finds its homologous partner, it pays for repair. 543 01:13:01,910 --> 01:13:05,690 But the problem gets a little bit more complex inside the cell because inside the 544 01:13:05,690 --> 01:13:09,680 cell you've got chromosome organisation chromosome compaction in several bacteria. 545 01:13:09,680 --> 01:13:13,220 You also have chromosome tethering of origin regions, for example, 546 01:13:13,220 --> 01:13:20,060 which can limit how much your your broken piece of DNA can move inside the cell to search for its homologous partner. 547 01:13:20,060 --> 01:13:24,410 So this form of constraint sort of led to the idea that maybe you don't search across 548 01:13:24,410 --> 01:13:29,180 the cell randomly and you search more in the vicinity of where the break occurred, 549 01:13:29,180 --> 01:13:35,390 and the search can be controlled than by how the chromosomes are organised or settled inside the cell. 550 01:13:35,390 --> 01:13:42,800 And so the idea then, is that maybe cells generally tend to engage in recombination in the polls that replicate of cohesion period in bacteria, 551 01:13:42,800 --> 01:13:50,300 and that once you segregate longer distances, it's less likely that you'll be able to do efficient homologous recombination. 552 01:13:50,300 --> 01:13:55,760 So really beautiful work by Christian Lesterland when he was in Deep Shut, its lab showed that that may not be the case. 553 01:13:55,760 --> 01:14:01,390 That. You can engage in sales, can engage in long distance search and do the combination in equally. 554 01:14:01,390 --> 01:14:05,110 And this involves the formation of a long recce nuclear power in filament, 555 01:14:05,110 --> 01:14:10,600 a bundle that spans sometimes the entire length of the cell that somehow manages to orchestrate homology. 556 01:14:10,600 --> 01:14:14,990 Such I'm following this in my own postdoc lab with my lab. 557 01:14:14,990 --> 01:14:18,910 I establish that even in colour back so that you have chromosomal tethering, 558 01:14:18,910 --> 01:14:24,400 segregated pieces of DNA can engage in long distance, homology, search and recombination. 559 01:14:24,400 --> 01:14:30,550 So then this still leaves open the question how inside the cell then does a broken piece of DNA sample 560 01:14:30,550 --> 01:14:37,540 several mega bases of DNA and compacted chromosomal environments to find a homologous partner efficiently? 561 01:14:37,540 --> 01:14:40,810 And so this is the question we set out to address in my lab. 562 01:14:40,810 --> 01:14:47,500 How does the recce filament orchestrate homology search and and they use call-back to consensus as a model system because all 563 01:14:47,500 --> 01:14:56,260 back has this very neat genetic trick that allows you to segregate or separate out cells in specific chromosomal content states. 564 01:14:56,260 --> 01:15:00,620 For example, we can separate out these these type of cells with a single mom replicating chromosome. 565 01:15:00,620 --> 01:15:04,630 We call these cuomos, or we can segregate separate out these type of cells. 566 01:15:04,630 --> 01:15:08,860 These three additional cells are two completely replicated and segregated chromosomes. 567 01:15:08,860 --> 01:15:14,500 So now in these cell types, we can ask about recombination or homology search alone without the confounding 568 01:15:14,500 --> 01:15:20,920 effects of overlapping replication cycles or ongoing replication during this process. 569 01:15:20,920 --> 01:15:28,600 So how do we induce a break? We use the IOC. One system to do that means that an IOC one site near the origin of the application and follow backed up, 570 01:15:28,600 --> 01:15:35,350 and we mark the site using an origin proximal marco ibs-c and then be induced to break while inducible expression 571 01:15:35,350 --> 01:15:41,200 of the IOC one enzyme such that we got one of the two chromosome in these cells and one chromosome in the cell. 572 01:15:41,200 --> 01:15:48,070 And using this system, we've extensively characterised the key steps of homologous recombination. 573 01:15:48,070 --> 01:15:52,960 We can now establish this the steps where lekin nucleus and loads at the double strand breaks. 574 01:15:52,960 --> 01:15:59,290 We can then look at homology so specifically and then look at homology pairing, followed by repair events. 575 01:15:59,290 --> 01:16:02,950 And I'm not going to go into these details because these are quite well established already. 576 01:16:02,950 --> 01:16:12,130 What I'm going to do today is focus on the part of homology search and try to understand how these dynamics play out inside the cell. 577 01:16:12,130 --> 01:16:20,080 And I want to highlight already that this work is has been led by one of my first graduate students, an exceptional graduate student of. 578 01:16:20,080 --> 01:16:25,150 And we work very closely with the theoretical physicist on campus mother and Rao. 579 01:16:25,150 --> 01:16:31,540 And I also thank already all funding agencies here, and we have a preprint of this work that was posted just a month ago. 580 01:16:31,540 --> 01:16:36,280 So please don't have a read when you get a chance. So what are the pros doing? 581 01:16:36,280 --> 01:16:40,540 So I decided to ask about the dynamics of recce in these solar cells, 582 01:16:40,540 --> 01:16:46,390 either in these cells with two chromosomes so we can look at search and repair or in cells with just a single chromosome, 583 01:16:46,390 --> 01:16:48,700 so we can look at just the search process alone. 584 01:16:48,700 --> 01:16:55,120 And she specifically looked at two parts of the rocky film, and dynamix one is where is the filament inside the cell? 585 01:16:55,120 --> 01:17:01,540 And second, how long is the filament so we can we can measure whether it's elongated or a short film. 586 01:17:01,540 --> 01:17:07,210 And so what approach does then is to track Ricky when she makes a break in these cells? 587 01:17:07,210 --> 01:17:11,320 So I'm going to show you her a movie of like with the image every one minute. 588 01:17:11,320 --> 01:17:17,320 But this wasn't done experiments where she's image every five seconds at incremental, at increasing intervals as well. 589 01:17:17,320 --> 01:17:21,190 So what are you going to see here is a break that has been made of this cell pole of the cell. 590 01:17:21,190 --> 01:17:27,760 And Ricky has already started sort of sort of started a nucleus of the cell phone and we're going to now track Ricky over time. 591 01:17:27,760 --> 01:17:32,440 And what we find is that when we image Ricky, it actually moves inside the cell. 592 01:17:32,440 --> 01:17:37,510 So it does form long structures, but it's dynamic. It doesn't stay in one position. 593 01:17:37,510 --> 01:17:42,580 It's actually moving in a very directional manner, from one cell phone to the opposite cell phones. 594 01:17:42,580 --> 01:17:47,470 And once it reaches the opposite cell pole, it reverses and then goes back in the same direction. 595 01:17:47,470 --> 01:17:50,590 And this is quite clear now with this very fluorescence profile. 596 01:17:50,590 --> 01:17:56,620 So what you see here is Ricky starting at one cell pole, it moves in a directional manner to the opposite cell Paul turns around, 597 01:17:56,620 --> 01:18:03,010 goes down again and does this iterative cycling multiple times before it finds its homologous partner. 598 01:18:03,010 --> 01:18:07,690 So Frost then characterises this traversal event by defining a reversal as Ricky, 599 01:18:07,690 --> 01:18:11,290 moving from one poll to the other cell phone and asked how long it takes. 600 01:18:11,290 --> 01:18:17,500 And we get, we see that on average, it takes about 300 to 400 seconds for it to go from one poll to the other, 601 01:18:17,500 --> 01:18:22,000 and it takes about six to eight cycles directly, finally finds its homologous partner. 602 01:18:22,000 --> 01:18:28,930 Of course, sometimes the partner can be found in one cycle itself, but on average it gets there by between six to eight cycles. 603 01:18:28,930 --> 01:18:33,220 So then the obvious question now is how is that moving directionally? 604 01:18:33,220 --> 01:18:40,990 Does it know where the homologous partner is? So this was an easy experiment for us to do, as they would say, a Friday afternoon experiment. 605 01:18:40,990 --> 01:18:46,600 She asked, Is that a homologous partner required for this directional movement? 606 01:18:46,600 --> 01:18:50,860 And so she conducted these experiments in the swarm of cells with just a single chromosome now. 607 01:18:50,860 --> 01:18:56,660 And what we find is that even in this case, they can move in a directional manner, and it never stopped. 608 01:18:56,660 --> 01:19:01,730 So when it reaches the opposite cell, Paul, it turns around and just keeps going back and forth because there's no thought, 609 01:19:01,730 --> 01:19:07,130 no threat to the pelvis and it's almost heart breaking, right? Because it just keeps touching. 610 01:19:07,130 --> 01:19:11,960 So clearly a partner attempted is not required for this movement, 611 01:19:11,960 --> 01:19:18,530 and it's something inherent about the property of the likely filament itself that's allowing it to move in this direction. 612 01:19:18,530 --> 01:19:21,320 So what is driving that movement? 613 01:19:21,320 --> 01:19:30,470 Now we know from really nice work done over several years that Recchia itself is unlikely to be a model so it can drive large scale displacements, 614 01:19:30,470 --> 01:19:38,090 but only be a cycle. We can envision in a cycle that allows for smaller growth and shrinkage of the a filament, 615 01:19:38,090 --> 01:19:42,800 but it's hard to see how it moves across long distances inside the cells. 616 01:19:42,800 --> 01:19:49,040 And so we wondered then what is causing this movement? So we did see that wreckage from several structures inside the cell. 617 01:19:49,040 --> 01:19:56,990 It can form a puncture. It can form an elongated filament that's turning at the pole or elongated filament inside the cell also. 618 01:19:56,990 --> 01:20:02,810 And there's no positional or spatial preference for what type of braking you see based on bed in the cell, 619 01:20:02,810 --> 01:20:10,400 so that it does toggle between various structures. But it's not just toggling between various structures during this process, it's also moving. 620 01:20:10,400 --> 01:20:17,900 So something is allowing for wreckage to not just change in its organisation about filament organisation, 621 01:20:17,900 --> 01:20:23,900 but it's also lot something is allowing the key to move inside the cells. And so the question becomes, What is that something? 622 01:20:23,900 --> 01:20:27,050 And we wondered, maybe we're thinking about this in an incomplete manner. 623 01:20:27,050 --> 01:20:33,110 Maybe it's not just working alone and there's something else involved with it is allowing for this movement to occur. 624 01:20:33,110 --> 01:20:37,580 And so what is then that mystery protein that allows for this movement? 625 01:20:37,580 --> 01:20:41,060 So we went after, I would say, my favourite protein. 626 01:20:41,060 --> 01:20:45,860 We went after them SNC protein rich and not much beef in this case. 627 01:20:45,860 --> 01:20:49,910 Second is a repair specific, some key protein in bacteria. 628 01:20:49,910 --> 01:20:56,450 We know that retcon physically interacts with backing from other bacterial systems, and we also know that it is essential for double strand break, 629 01:20:56,450 --> 01:21:02,270 repair and utilise cells knocked out for can sensitivity to blue myosin, for example. 630 01:21:02,270 --> 01:21:09,740 Even in our own setup, when we ask how many cells are able to do long distance search and repair, we find that in the absence of lichen, 631 01:21:09,740 --> 01:21:17,300 we cannot see the bad events in long distance, which suggests then that is essential for long distance search. 632 01:21:17,300 --> 01:21:23,060 So then we ask the question What happens to Lecky Dynamics if you don't have that in the cells? 633 01:21:23,060 --> 01:21:27,470 So it shows that these experiments and I'm going to show you a movie that took every five minutes. 634 01:21:27,470 --> 01:21:33,020 And as we become clear to you why I'm showing you such long time scale movie, she has done shorter time scale movies also. 635 01:21:33,020 --> 01:21:40,100 What we find is that in addition, deletion recce forms at the break site, but it does not move. 636 01:21:40,100 --> 01:21:45,500 You see a nice filament forming, but you don't see long distance reversal inside the cells. 637 01:21:45,500 --> 01:21:51,890 And this gets even more highlighted when you look at the recce fluorescence profile and point you out to the x axis. 638 01:21:51,890 --> 01:21:59,510 It's going to more than 100 minutes, that confirms, but it does not move after forming at the break sites. 639 01:21:59,510 --> 01:22:08,540 And so just to just to highlight again the comparison when you look at several such recce localisation events over the course of imaging, 640 01:22:08,540 --> 01:22:15,290 we do not see any movement at all. But as I showed you in the wild Typekit, you see this very nice movement of Lecky in a directional manner, 641 01:22:15,290 --> 01:22:19,640 from one cell to the other within a matter of a few hundred seconds. 642 01:22:19,640 --> 01:22:24,590 And this holds true irrespective of whether you have a template for repeat or not. 643 01:22:24,590 --> 01:22:33,770 This is again simply a combination of the property of Lecky and retconned together that enables for for this movement to occur. 644 01:22:33,770 --> 01:22:44,580 And so we wonder then what is it about Rackham that is allowing for for the Leket movement, this large scale movement of Lecky inside the cells? 645 01:22:44,580 --> 01:22:49,870 So we went off and sort of ruled out, you know, only early, 646 01:22:49,870 --> 01:22:56,310 though the more obvious suspects supplicant could be affecting the ask does it affect the rate at which nuclear to the black side? 647 01:22:56,310 --> 01:23:01,950 And we found that it does not affect the rate at which technically it's the bleak side. We then ask, does it affect the later recce? 648 01:23:01,950 --> 01:23:06,180 Filament grows once it is formed at a break, and it does not either. 649 01:23:06,180 --> 01:23:09,480 And then we finally ask, does it affect the filament stability of record? 650 01:23:09,480 --> 01:23:14,640 So we know that in cells that don't have recce, cells can undergo something called reckless degradation, 651 01:23:14,640 --> 01:23:20,490 where they degrade extensively or extensively DNA around the black site. 652 01:23:20,490 --> 01:23:21,750 In the absence of Lekin, though, 653 01:23:21,750 --> 01:23:29,820 you don't get extensive dissection to suggest that it is binding just fine at the at the break site and protecting the DNA from excessive degradation. 654 01:23:29,820 --> 01:23:34,710 So we don't doubt out key filament stability as a problem as well. 655 01:23:34,710 --> 01:23:42,000 So here we are, right? So what I've shown you is that you can can localise at a side once there is a double spring break, 656 01:23:42,000 --> 01:23:50,730 but it's the presence of Recon that allows it to do this long distance movement to then do homology, search of homology, search and repair. 657 01:23:50,730 --> 01:23:58,080 And we know that this movement is essential for long distance recombination because in the absence of lichen, you get no repair at all. 658 01:23:58,080 --> 01:24:03,660 So clearly this the step is enabling Ricky to do homology search inside the cell. 659 01:24:03,660 --> 01:24:11,640 And so then what is the dynamic mechanism by which lichen is driving such rocky filament remodelling and translocation in the cell? 660 01:24:11,640 --> 01:24:16,260 And so we were at this point of the project and we were wondering how can we understand the 661 01:24:16,260 --> 01:24:21,300 mechanism better when we started talking to a theoretical physicist on campus modern ro? 662 01:24:21,300 --> 01:24:27,330 What things about cytoskeleton elements? A lot in eukaryotic systems or microtubule dynamics, for example? 663 01:24:27,330 --> 01:24:31,260 And he looked at our data and he said, Look, maybe there's two ways of thinking about it. 664 01:24:31,260 --> 01:24:36,870 One way of thinking about the effect of lichen is while something like motor driven translocation, 665 01:24:36,870 --> 01:24:43,260 it's possible that retcon is a motor that's driving this long-distance movement of Ricky. 666 01:24:43,260 --> 01:24:49,850 But one one prediction of modern urban translocation would be that you would see a static filament moving inside the cell, 667 01:24:49,850 --> 01:24:55,200 and you wouldn't see large fluctuations in filament length as it's moving inside the cell. 668 01:24:55,200 --> 01:25:00,390 But we do see that. So if you look at the changes in filament length, so this is what we call Delta L. 669 01:25:00,390 --> 01:25:08,040 You see that there is lot larger fluctuations in the lengths of Lecky in the wild type cells, but you don't see so much interaction deletion. 670 01:25:08,040 --> 01:25:14,060 So motor driven translocation alone cannot explain the movement that we're observing inside the cell. 671 01:25:14,060 --> 01:25:18,530 So the other way of thinking about it, then, is perhaps it's affecting assembly this assembly, 672 01:25:18,530 --> 01:25:22,970 so maybe that kind of somehow controlling the rate at which it assembled or disassembled 673 01:25:22,970 --> 01:25:28,130 on a single strand of DNA that then perhaps drives movement inside the cell? 674 01:25:28,130 --> 01:25:32,090 And so Mullen looked at these dynamics specifically, 675 01:25:32,090 --> 01:25:39,170 so he mapped out binding UN binding plates of Lekki on single strand of DNA in the presence or absence of action, 676 01:25:39,170 --> 01:25:44,630 and asked Would there be a scenario where one could explain the binding and on binding rates of 677 01:25:44,630 --> 01:25:49,760 Lekki that are dependent on wrecking that allows for net positive movement in one direction? 678 01:25:49,760 --> 01:25:56,360 And he was able to theorise such a scenario based on the fact that we have a stable length of Lekki finally. 679 01:25:56,360 --> 01:26:00,650 So if you look at the mean length of Lekki, it never extends across the whole length of the cell. 680 01:26:00,650 --> 01:26:05,990 It actually has a fixed mean length that is, you know, around 0.2 percent of the wholesale length, 681 01:26:05,990 --> 01:26:10,190 which is about one micron in a three to four micron long cell. 682 01:26:10,190 --> 01:26:14,780 And so based on this, you said, OK, I can explain all of these dynamics with this equation, 683 01:26:14,780 --> 01:26:19,310 and solving this equation gives you a distribution of lengths that you predict for Ricky 684 01:26:19,310 --> 01:26:25,640 inside the cell that very closely match what we see already in our in our experimental setup. 685 01:26:25,640 --> 01:26:30,140 So this suggests that definitely reckoned is affecting the length of Lekki directly. 686 01:26:30,140 --> 01:26:36,860 Another test for whether Akon is affecting the length of Lekki was the turn to the Recon Apes cycle itself. 687 01:26:36,860 --> 01:26:41,870 So we asked whether the ATP cycle plays any role in the Lekki dynamics we observed. 688 01:26:41,870 --> 01:26:49,070 So there can be an assembly protein has concealed carry and what could be residues that participate in ATP binding and hydrolysis. 689 01:26:49,070 --> 01:26:57,320 And so we mutated these residues specifically and ask them, what effects does this have on breaking movement as well as wrecking filament lengths? 690 01:26:57,320 --> 01:27:01,640 And so we made two mutants who made a mutant added to a mutant that is thought to 691 01:27:01,640 --> 01:27:06,410 affect ATP binding and an idoko mutant that is thought to affect ATP hydrolysis, 692 01:27:06,410 --> 01:27:08,750 although not completely, but somewhat. 693 01:27:08,750 --> 01:27:14,660 And in both cases, we got static filaments that does not move inside the cell with the structure of these filaments with the opposite. 694 01:27:14,660 --> 01:27:22,520 So in the ATP binding mutant, yes, we saw longer filaments, but in the hydrolysis mutant, we saw these very tight functional structures. 695 01:27:22,520 --> 01:27:25,670 We do not sample longer filaments inside the cells, 696 01:27:25,670 --> 01:27:31,910 suggesting that the Ricken ATP a cycle is actually affecting how long or small decayed filaments can be. 697 01:27:31,910 --> 01:27:40,790 And given that the filament does not move in either scenario, this length fluctuation is likely important for Ricky to move inside the cell. 698 01:27:40,790 --> 01:27:46,850 Right, of course. I mean, we still can't rule out water driven translocation because they can use an assembly family feuding, 699 01:27:46,850 --> 01:27:54,740 and there is ample evidence now to suggest that some cities do engage in some form of motor driven translocation by a loop exclusion, for example. 700 01:27:54,740 --> 01:27:59,660 So we don't need to do a lot more experiments to test to see whether we can rule in or rule out this. 701 01:27:59,660 --> 01:28:05,120 In fact, it's possible that it's a combination of both, and that's how we're envisioning this whole scenario right now. 702 01:28:05,120 --> 01:28:08,660 So in some ways, to summarise what I've what I've sort of shared with you so far, 703 01:28:08,660 --> 01:28:14,990 I've shown you that when there is a double spend break and pull it back to cells on the call back to chromosome, 704 01:28:14,990 --> 01:28:20,450 it results in the new creation of molecular nuclear power in filament that is quite dynamic inside the cell. 705 01:28:20,450 --> 01:28:26,900 And this dynamic nuclear protein filament undergoes two things it undergoes dynamic remodelling of its architecture, 706 01:28:26,900 --> 01:28:31,070 but it also undergoes iterative movement inside the cell in the direction of metals. 707 01:28:31,070 --> 01:28:34,370 It goes all the poles searching for its homologues partner, 708 01:28:34,370 --> 01:28:43,250 and we like now envision this movement as a form of ligand driven recce motion via controlling how quickly it associates or 709 01:28:43,250 --> 01:28:51,200 dissociate from the DNA substrate in conjunction with retcons sort of moving the DNA substrate itself along in the cell. 710 01:28:51,200 --> 01:28:54,290 And this could be coming by our exclusion as other assembly proteins do, 711 01:28:54,290 --> 01:28:57,680 or even by zipping with with retcon sort of zipping together the single and the 712 01:28:57,680 --> 01:29:03,230 double standard DNA together as it moves inside the cell to do homology search. 713 01:29:03,230 --> 01:29:06,890 So but what does it mean for homology search itself if you can do it this way? 714 01:29:06,890 --> 01:29:14,480 Right. So the idea of having a reception or iteration built into such allows you to search in a more 715 01:29:14,480 --> 01:29:19,550 efficient manner because it allows you to have a fixed position to which you can come back to. 716 01:29:19,550 --> 01:29:26,600 And so now suddenly you are not dependent on on geometry. If you if you're abroad, you can go in, you're in the long axis. 717 01:29:26,600 --> 01:29:32,540 But if you are a circle, you can go in any axis and come back to your fixed point again and then start the process again. 718 01:29:32,540 --> 01:29:38,240 And so this idea of searching is a lot more efficient than random sampling inside the cell. 719 01:29:38,240 --> 01:29:40,940 And it also doesn't depend on your genome getting mobilised. 720 01:29:40,940 --> 01:29:50,750 You just need your broken piece of DNA to move inside the cells and engage in microbiology sampling as it's moving during the search process. 721 01:29:50,750 --> 01:29:54,440 Right. So this is where we are right now, and this is where we want to go next. 722 01:29:54,440 --> 01:29:58,430 We want to try and figure out what is the molecular mechanism by which Lekin is driving the 723 01:29:58,430 --> 01:30:04,430 key filament dynamics and movement and how important is the position of the black side. 724 01:30:04,430 --> 01:30:12,350 And in driving these dynamics are shown you today. OK, so that's the story, and I already thank the people involved, 725 01:30:12,350 --> 01:30:18,590 so I'm going to shift gears a little bit and thank the person we are here to celebrate today. 726 01:30:18,590 --> 01:30:27,380 So I met Dave, but I had come to Oxford for my master's degree and I met him for a master's thesis project. 727 01:30:27,380 --> 01:30:31,610 And this was the first time I I experienced this question. 728 01:30:31,610 --> 01:30:37,310 I had never thought of this problem before that the cell has a lot of DNA and that DNA 729 01:30:37,310 --> 01:30:43,010 has to somehow fit inside the cell in an organised manner and be used by the cells. 730 01:30:43,010 --> 01:30:50,600 And then when they've explained this problem to me and the slide is quite striking, then Donald shared it with Ted, the slide with me. 731 01:30:50,600 --> 01:30:57,290 And this is a slide that Dave has also used several times to try and illustrate the the issue that the cell is dealing with. 732 01:30:57,290 --> 01:31:01,910 How do you take a chromosome and compact a thousand fold to fit inside the cell? 733 01:31:01,910 --> 01:31:09,710 And this was such a fascinating problem, and I am just so grateful that I had the chance to to engage with this problem and try to 734 01:31:09,710 --> 01:31:16,590 understand how cells do chromosome organisation and compact condensation during this process. 735 01:31:16,590 --> 01:31:23,270 I also learnt that it's not just about chromosome organisation, it's also about chromosome segregation. 736 01:31:23,270 --> 01:31:29,210 It's also about maintaining genomes via DNA repair or recombination. 737 01:31:29,210 --> 01:31:34,970 It's also about doing this in conjunction with activities such as transcription and this 738 01:31:34,970 --> 01:31:41,360 sort of holistic picture of of what it means to to maintain chromosomes inside the cell. 739 01:31:41,360 --> 01:31:49,910 Came in Dave's lab for me, and I am so grateful for that and for the people who allowed me to understand that it's not just one problem, 740 01:31:49,910 --> 01:31:54,110 it's actually many problems together and that all of them have to work together 741 01:31:54,110 --> 01:32:00,020 to allow for a cell to then faithfully propagate life over several generations. 742 01:32:00,020 --> 01:32:06,380 And so I want to thank all of these people too far for sort of opening for me, 743 01:32:06,380 --> 01:32:11,640 the the idea and the understanding of what it means to have a chromosome inside of cells. 744 01:32:11,640 --> 01:32:22,700 And thank you, Dave, for for bringing everyone together and for being so enthusiastic and taking us through this journey of the cells chromosome. 745 01:32:22,700 --> 01:32:30,020 In some ways, I want to I want to sort of replace again and say that, you know, many of us, at least me, I can do for myself. 746 01:32:30,020 --> 01:32:34,700 I come to Dave like Ricky without direction, and there's Dave Shadid's, 747 01:32:34,700 --> 01:32:39,980 who comes in like a cool vein and says, I let you search and let you search for. 748 01:32:39,980 --> 01:32:43,040 Don't answer. But I'm going to guide how you search. 749 01:32:43,040 --> 01:32:53,380 And that's so efficient, and it's been such and such a beautiful journey to experience science with this amazing, amazing person. 750 01:32:53,380 --> 01:32:57,770 And I want to also highlight one thing that I think many people sort of touched upon. 751 01:32:57,770 --> 01:33:05,120 But I want to specifically say I think they've had a great way of bringing people together and giving them an 752 01:33:05,120 --> 01:33:12,950 environment where they can be happy to do science and engage in science for the sake of science driven by curiosity, 753 01:33:12,950 --> 01:33:15,950 driven by by the fun of discovery. 754 01:33:15,950 --> 01:33:22,910 And you see such happy faces in all these pictures because I think it's the environment created for all of us to engage together, 755 01:33:22,910 --> 01:33:29,540 to collaborate together and to answer these fundamental questions in biology. 756 01:33:29,540 --> 01:33:31,970 This doesn't stop doing that. 757 01:33:31,970 --> 01:33:41,360 He joined lab meeting with With My Lab last year, and so thank you, Dave, for spreading your enthusiasm to people here as well. 758 01:33:41,360 --> 01:33:45,800 I think that is just so beautiful that you take the time to do that. 759 01:33:45,800 --> 01:33:57,860 And this is a picture of my graduation with my family and extended family at Oxford just before I left for my postdoc with my lab at MIT. 760 01:33:57,860 --> 01:34:05,990 I'll stop here with this picture that my postdoc Asia had taken at a farm summer school in 2019, 761 01:34:05,990 --> 01:34:15,650 where Davis was there in his characteristic form, with his tube around his neck there to explain how topology works. 762 01:34:15,650 --> 01:34:22,070 And he he again said the same thing that he has always said this is his words from an 763 01:34:22,070 --> 01:34:27,320 interview he had done with a phenotype the Oxford University Biochemical Society Journal. 764 01:34:27,320 --> 01:34:35,030 They talked about how important it is to be passionate and just go and answer questions without being afraid. 765 01:34:35,030 --> 01:34:40,220 So keeping your legacy on the I'm sure all of us are going to go ahead and do just that. 766 01:34:40,220 --> 01:34:52,000 Thank you. And at this time, I'll take questions. 767 01:34:52,000 --> 01:35:00,280 Thank you. I, Anjana, can you hear me? 768 01:35:00,280 --> 01:35:04,970 Hey, Dave, I can hear you. Yes. Lovely to hear from you. 769 01:35:04,970 --> 01:35:17,840 Revkin seems to be an outlier in the sense that every other sea protein that I know of works as a complex with accessory proteins, Gleeson's whatever. 770 01:35:17,840 --> 01:35:23,830 Do you have any knowledge or feeling as to whether Revkin is autonomous? 771 01:35:23,830 --> 01:35:26,260 Yeah, I know that's that's a really good question, we don't know, 772 01:35:26,260 --> 01:35:32,530 so we are trying to set up pull downs right now to try and see what else they can associates with. 773 01:35:32,530 --> 01:35:40,630 We know that E! Affects reckons ATPase activity, so it's possible that that accessory role has been taken up by Ricky instead. 774 01:35:40,630 --> 01:35:46,090 But I mean, it's a good question. We can't rule it out unless we try to see what else they can interacts with. 775 01:35:46,090 --> 01:35:56,260 We don't know the answer to that. High end journalist, I'm over on the side here, and I'm sure you seen as Steve Kornacki so very, very high. 776 01:35:56,260 --> 01:36:01,490 Hi, thanks for saying pre-print. Very nice, very provocative data. 777 01:36:01,490 --> 01:36:04,670 And you know, I took a comment and a question. 778 01:36:04,670 --> 01:36:12,090 So the comment is, you know, the one dimensional search, which is really what you're saying, even though it's directional, is very inefficient. 779 01:36:12,090 --> 01:36:18,980 I mean, you even had that nice chromosome spread at the end of your talk and showed how much DNA you'd have to traverse to go across. 780 01:36:18,980 --> 01:36:23,000 I mean, in terms of it being looped and folded, and we heard talks about that. 781 01:36:23,000 --> 01:36:29,300 So following along that track is is it an is a very fast motor, which most species are not? 782 01:36:29,300 --> 01:36:33,890 And again, I know that's controversial too. But but it just doesn't. 783 01:36:33,890 --> 01:36:39,050 I'll say it doesn't make sense to me. So, but you know, it's the data are the data are beautiful. 784 01:36:39,050 --> 01:36:45,290 So the question for you is I was struck by the movements being much like this par ab system. 785 01:36:45,290 --> 01:36:51,450 And I know there's another system behaves like I can't remember which won the pole to pole move and oscillation. 786 01:36:51,450 --> 01:36:55,520 It just almost seems like the PA system. 787 01:36:55,520 --> 01:36:59,960 So I'm trying to figure out. Yes, you're a and observation is indeed correct. 788 01:36:59,960 --> 01:37:06,050 But does is wreck in fact moving along with some other component that's not tracking the DNA, 789 01:37:06,050 --> 01:37:15,860 but rather somehow moving along some cytoskeleton holes or membrane part, something that's not a long, skinny linear track. 790 01:37:15,860 --> 01:37:20,450 So can you can you have you thought about that and just commented on it? 791 01:37:20,450 --> 01:37:22,560 Yeah, no, that that's a really nice questions. I mean, 792 01:37:22,560 --> 01:37:31,940 we thought of in the context of a because there were these mutants that people had isolated in equally showing that it can associated the membrane. 793 01:37:31,940 --> 01:37:34,970 If you make these mutants, you can break membrane association. 794 01:37:34,970 --> 01:37:42,830 These are not cousins of mutants, and we did try to make mutations in similar areas with Becky, but we didn't see any effect on wrecking movement. 795 01:37:42,830 --> 01:37:45,650 So we didn't think of it in the context of that kind is possible. 796 01:37:45,650 --> 01:37:53,000 Maybe like is associating with cytoskeleton element, maybe MRSA or something that then drives that is acting as a trap. 797 01:37:53,000 --> 01:37:57,320 But just to sort of counter your idea of just pure oscillation, you know, 798 01:37:57,320 --> 01:38:02,510 because if it was just oscillation, one would see sort of linear tracts of the rocky filament. 799 01:38:02,510 --> 01:38:07,970 And maybe if there is time for one movie, I'll play a movie. 800 01:38:07,970 --> 01:38:12,020 If that's OK, we have maybe a minute or half a minute. So that's fine. 801 01:38:12,020 --> 01:38:16,850 OK, I'm going to then we'll play a movie before the answer comes OK. All right. 802 01:38:16,850 --> 01:38:26,720 So if you if you see these movies, you can actually see that the filament is changing quite a bit in its in its shape, 803 01:38:26,720 --> 01:38:31,820 and it's possible that it's actually popping across larger segments of DNA. 804 01:38:31,820 --> 01:38:35,240 And so that can allow for for longer movements, right? 805 01:38:35,240 --> 01:38:38,870 And so it may not be just one d sliding. 806 01:38:38,870 --> 01:38:47,840 It could also be hopping across segments of DNA, you know, maybe even domains of DNA, and that allows them for it to jump across a lot more. 807 01:38:47,840 --> 01:38:56,530 Of course, this needs to be tested. I can't we don't know whether this is really the case, but this is a possibility also. 808 01:38:56,530 --> 01:39:00,790 There. High end diner. 809 01:39:00,790 --> 01:39:07,900 And that was that was really fantastic from from your agency, detective agency outpost. 810 01:39:07,900 --> 01:39:15,010 So I was wondering as in there, I can meet and you still got sort of quite extended filament, 811 01:39:15,010 --> 01:39:20,010 yet you said that they're the commodities such as basically not happen anymore. 812 01:39:20,010 --> 01:39:21,820 There's no right recombination happening. 813 01:39:21,820 --> 01:39:27,670 And that's somewhat surprising that eventually you would think maybe of a long time the right segments would come together. 814 01:39:27,670 --> 01:39:32,350 So this is maybe the role of rock n rather to take off the sort of the wrong bits 815 01:39:32,350 --> 01:39:36,910 of the DNA doesn't prevent the random access of other of other segments of DNA, 816 01:39:36,910 --> 01:39:42,440 and that's that can help to to get the right filament off of the wrong place, basically. 817 01:39:42,440 --> 01:39:45,960 Yeah. Yeah, yeah, yeah. That's that's a nice way of thinking about it. 818 01:39:45,960 --> 01:39:47,830 So I want to answer the question in two parts. 819 01:39:47,830 --> 01:39:55,990 The first part is that Ricky never agrees to called back to I can never make the long enough filament that goes from one poll to the other. 820 01:39:55,990 --> 01:40:00,550 The filament is always less than one quarter of the cell. 821 01:40:00,550 --> 01:40:10,120 And so in that sense, for long distance search, the filament would never become long enough to reach the other end, but it can do the best. 822 01:40:10,120 --> 01:40:15,730 So that's perhaps one reason why we don't see the pattern the can deletion at some point, right? 823 01:40:15,730 --> 01:40:21,160 But it's also possible that Rickon is regulating decades sand invasion capability so 824 01:40:21,160 --> 01:40:25,420 that these two pieces of evidence of that are quite interesting and quite far away. 825 01:40:25,420 --> 01:40:31,510 But so one one piece of evidence is that Rick and stimulates decades IDPs cycle. 826 01:40:31,510 --> 01:40:36,970 And then the other piece of evidence separately is that the sand invasion been been decades doing microbiology. 827 01:40:36,970 --> 01:40:46,090 Sampling also stimulates APIs of Ricky. And so it's it's possible that reckoning is bringing together double standard and single strand 828 01:40:46,090 --> 01:40:51,730 of DNA to stimulate strand invasion and that that allows turnover of Ricky more rapidly. 829 01:40:51,730 --> 01:40:55,120 Again, all of this needs to be tested admissible in vitro and they have. 830 01:40:55,120 --> 01:41:03,460 You didn't teach me how to purify protein, so we need to learn that the hard way now. 831 01:41:03,460 --> 01:41:10,690 And John, I came here as lovely talk. Sorry, sorry, you can't be here. 832 01:41:10,690 --> 01:41:17,980 The I had a couple of questions. One is in your system, you're breaking. 833 01:41:17,980 --> 01:41:25,990 You know, when you had to, both the donor and the recipient have both broken potentially is because idea. 834 01:41:25,990 --> 01:41:32,500 And this is a problem with a lot of these systems. The same problem with the whole system was always a sort of criticism of those is that 835 01:41:32,500 --> 01:41:37,570 you really want to break one and not and then have an intact partner to be found. 836 01:41:37,570 --> 01:41:44,680 I don't know whether that you can make any progress on that. My other question is, have you actually been able to image reckon? 837 01:41:44,680 --> 01:41:49,960 And you know, what does it does? Does it track with Rick or is it? 838 01:41:49,960 --> 01:41:54,400 I mean, has that been has that revealed any insight? OK, great. 839 01:41:54,400 --> 01:41:59,380 So for the first question, we maybe have a cheap mic mechanism, 840 01:41:59,380 --> 01:42:05,860 so we essentially only look at cells where one of the two brick markers are lost in cells, 841 01:42:05,860 --> 01:42:10,870 where we lose both the black site markers, we don't regard them because now we know both the sites are broken. 842 01:42:10,870 --> 01:42:15,700 So we only go after the cells with with a single single break, Malcolm lost. 843 01:42:15,700 --> 01:42:22,000 So that's why we are able to look only at the record at single break, followed by recombination events. 844 01:42:22,000 --> 01:42:28,810 But for the second question, we did look at attraction. I mean, these are very preliminary data and we have to analyse them better. 845 01:42:28,810 --> 01:42:34,510 So that can is actually quite dynamic. It always localises with Becky, and there are few rules. 846 01:42:34,510 --> 01:42:42,040 Silicon does not localise onto the break site without wreckage, so it comes after directly and it closes at the beginning. 847 01:42:42,040 --> 01:42:47,530 But then over time, during the search process, you can see looking at various parts of the decay filaments. 848 01:42:47,530 --> 01:42:50,920 Sometimes it's at the back end, sometimes it's at the front end, 849 01:42:50,920 --> 01:42:55,300 and sometimes it's coating the whole of the filament so that these three main categories, 850 01:42:55,300 --> 01:43:05,150 and we don't quite know as yet how each of these are perhaps leading to the other or or what each of these categories mean for Four-decade movement. 851 01:43:05,150 --> 01:43:10,740 So that's something they need to work out now. Keith, thank you very much. 852 01:43:10,740 --> 01:43:14,233 I'm Jenna for this night's a discussion, so.